Instrument for performing a diagnostic test on a fluidic cartridge

ABSTRACT

A cartridge reader is configured to carry out a diagnostic test on a fluid sample contained within a fluidic cartridge. The cartridge comprises first, second and third collapsible blisters containing at least one reagent for use in the diagnostic test. The cartridge reader comprises an upper clamp, occupying a fixed position relative to the reader and a lower clamp, movable relative to the upper clamp, and wherein the upper clamp and the lower clamp are configured to receive and hold a fluidic cartridge therebetween. First, second and third blister actuators are mounted on the upper clamp, for aligning with first, second and third collapsible blisters of a fluidic cartridge inserted into the reader. The first, second and third blister actuators are movable relative to the upper clamp, between a first position in which the blister actuators are spaced apart from the collapsible blisters comprised on the fluidic cartridge received between the upper and lower clamps, and a second position in which the blister actuators depress the collapsible blisters, thereby collapsing the blisters and ejecting the reagents contained therein into a channel in the microfluidic cartridge.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 15/547,786, filed Jul. 31, 2017, now U.S. Pat. No.10,751,719, which is a national stage application, filed under 35 U.S.C.§ 371, of PCT International Application No. PCT/GB2016/050231, filed onFeb. 2, 2016, and claims benefit of priority to British PatentApplication No. 1501704.9, filed on Feb. 2, 2015, each of which,including their contents, are incorporated herein by reference in theirentireties.

FIELD

The present invention relates to a cartridge reader for carrying out adiagnostic test on a fluid sample contained in a fluidic cartridge, andreading a result therefrom.

BACKGROUND

Sample preparation and analysis presents many logistical problems.Conventionally, many medical samples (such as blood, saliva, urine andswab eluate) are provided to a doctor, for example a generalpractitioner doctor (GP) or a principle care physician (PCP), in a localsurgery without the equipment necessary to analyse the sample. Hence,the sample must be sent to a laboratory where the sample is analysed.The test results must then be collated and returned to the GP to analysethe results and make a diagnosis. This approach is inadequate. Firstly,there is a significant risk that a sample is lost in transit ormismatched with the wrong patient. Moreover, whilst recent developmentsin technology have reduced the overall time taken to conduct the test,the delay involved in sending the sample to a laboratory isunsatisfactory.

Nevertheless, analytical systems of the kind found in laboratories arecomplex and it is often difficult to provide sufficient amounts of puretargets from source samples to reliably perform downstream analyticalassays. This typically prohibits local GP surgeries from being able tocarry out such tests on site.

However, in recent years efforts have been made to reduce the scale ofthe analytical systems to make tests faster and simpler to run, andrequire smaller quantities of sample. For instance, “laboratory on achip” (LOC) devices (a subset of microfluidic devices) integrate almostall medical tests or diagnostic operations performed in a hospital on asingle microfluidic chip. The channels forming such microfluidicsdevices handle small fluid volumes and are connected together so as toachieve a desired function such as mixing of a sample, moving the samplethrough the device, reacting the sample with different reagents, and soon. These chips may be inserted into machines to control the performanceof a test and measure the results.

However, it has been found that handling a sample in a microfluidicsdevice can be very difficult. In particular, it is difficult tointerface to the small channels and other features that are required tomove the sample from one site to another to perform different actions onthe sample. There is also a limit to the complexity of a LOC devicewhich operates purely using capillary action. Furthermore, owing to thesmall sample sizes of LOC's, the devices have reduced sensitivity andthe probability of a target being present in the sample is thus reduced.

An alternative approach is to use a fluidic cartridge. The scale of thecomponents of a fluidic cartridge is larger than for a microfluidicdevice, and so it becomes possible to move a sample through variousdifferent sites to perform different actions on it. This makes itpossible to perform more complex tests than may be conducted usingtypical LOC devices, whilst still providing an analytical system ofpotential use in a local GP surgery.

Fluidic cartridges are generally inserted into a cartridge readerconfigured to initiate and control at least some of the steps of a testto be carried out. For example, cartridge reader may initiate a test bydetecting the presence of a cartridge and moving a sample through thevarious channels in the cartridge. The reader may initiate theintroduction of required reagents into the cartridge, and controlvariables such as sample temperature throughout the duration of thetest. Finally, the reader may be configured to read and display a resultto the user, once the required test has been carried out.

Increasingly, scientific assays useful in medical diagnostics haveinvolved biochemical procedures, such as the polymerase chain reaction(“PCR”). The PCR assay has provided a particularly sensitive method ofassaying for the presence of defined segments of nucleic acids. It istherefore desirable to perform a PCR assay on a fluidic cartridge, andto provide a cartridge reader, suitable for use in a local surgery ofdoctors' office capable of carrying out and/or controlling a PCR assay.The use of PCR requires rapid and reliable thermal control on thecartridge,

Reducing PCR to the microchip level is important for portable detectiontechnologies and high throughput analytical systems. The method can beused to assay body fluids for the presence of nucleic acid specific forparticular pathogens, such as the Chlamydia trachomatis bacterium, HIVor any other pathogenic microbe.

The introduction of commercially available automated DNA amplificationassays has allowed more laboratories to introduce these technologies forroutine testing of specimens. However, there is a need to improve thecartridges and cartridge readers used for this purpose.

Electrochemical signalling may be used to indicate the presence ofgenetic or immunohistochemistry targets in a sample. The sample isprocessed to form an electrolyte which, in practice, may be held in acell comprising a set of detection electrodes. Upon application of apotential difference across electrodes in the cell, some compounds in anelectrolyte will have a natural tendency to migrate to the electrodesand swap electrons, resulting in a tiny current All combinations ofsoluble compounds have some electrochemical activity, and the rate atwhich this activity occurs enables measurement of the quantity of thosecompounds. Thus, the presence of different compounds in the sample maybe measured by searching for characteristic features of their redoxelectrochemistry. In particular, the sample may be processed to includelabels: selected compounds that are present if and only if the samplecontains target molecules.

A circuit used to measure electrochemical activity is a potentiostat,which has three electrodes—a working electrode, a counter electrode anda reference electrode. A potential difference is applied across theworking and counter electrodes and, as a result, a label indicating thepresence of target DNA oxidizes on the working electrode and a currentflows from the working electrode to the counter electrode. This currentis dependent of the galvanic activity (natural reactivity betweenelectrode and electrolyte) at both electrodes, and in order that onlythe effect of the working electrode electrochemistry is measured, thepotential difference applied is corrected for the galvanic activity ofthe counter electrode by an amount determined by the reference electrodewhich is chosen to be a ‘standard electrode’ relatively unchanged bylocal chemistry effect.

The current flowing at any given thus-corrected potential difference ismeasured and provides the signal that is indicative of the labelcompounds in the sample. Conventionally, the counter electrode isexcessively large so that the reaction at this electrode does not limitthe current flowing as a result of the reaction of the workingelectrode, which is the one of interest.

However, during development of a cartridge and a cartridge reader asdescribed above, the inventors found that the signals generated bypotentiostats of conventional design were unsatisfactory, and thus had aneed for an improved arrangement that generated better signals.

SUMMARY OF INVENTION

In a first aspect, the present invention provides a cartridge readerconfigured to carry out a diagnostic test on a fluid sample containedwithin a fluidic cartridge, wherein the cartridge comprises first,second and third collapsible blisters containing at least one reagentfor use in the diagnostic test, the cartridge reader comprising:

an upper clamp, occupying a fixed position relative to the reader and alower clamp, movable relative to the upper clamp, and wherein the upperclamp and the lower clamp are configured to receive and hold a fluidiccartridge therebetween;

first, second and third blister actuators mounted on the upper clamp,and for aligning with first, second and third collapsible blisters of afluidic cartridge inserted into the reader, wherein the first, secondand third blister actuators are movable relative to the upper clamp,between a first position in which the blister actuators are spaced apartfrom the collapsible blisters comprised on the fluidic cartridgereceived between the upper and lower clamps, and a second position inwhich the blister actuators depress the collapsible blisters, therebycollapsing the blisters and ejecting the reagents contained therein intoa channel in the microfluidic cartridge.

Preferably, the cartridge further comprises a recess, comprising amechanical valve for depressurising a region of the cartridge, the valvehaving first and second valve seats, wherein the first valve seatcomprises one port and the second valve seat comprises two ports, andfirst and second valve membrane portions configured, upon actuation, toseal the first and second valve seats, respectively, and wherein theupper clamp further comprises a mechanical valve actuation assembly foractuating the first and second valve membrane portions, the mechanicalvalve actuation assembly comprising:

a first actuator mechanism for actuating the first valve membraneportion and a second actuator mechanism for actuating the second valvemembrane portion;

wherein the first actuator mechanism is mounted to the upper clamp andis movable relative to the upper clamp between a first position in whichit is spaced apart from the first valve membrane portion and a secondposition in which it actuates the first valve membrane portion; andwherein the second actuator mechanism is mounted to the first actuatormechanism by a resilient biasing means and is movable relative to thefirst actuator mechanism, such that it is configured to actuate thesecond valve membrane portion before the first actuator mechanism ismoved to its second position.

Preferably, each of the first, second and third blister actuatorscomprises a housing portion from which extends a stem portion havingmounted on its end a tip portion for contacting the collapsibleblisters, wherein the tip portion of one or more of the first, secondand third blister actuators has an outwardly-extending curved profile.

Preferably, the tip portion of one or more of the first, second andthird blister actuators has a substantially flat profile.

Preferably, the first and second blister actuators has anoutwardly-extending curved profile and the third blister actuator has asubstantially flat profile.

Preferably the reader further comprises an actuator controller, andwherein each of the first, second and third blister actuators are linearactuators driven by a stepper motor, the actuator controller coupled toeach stepper motor and configured, upon actuation, to maintain each ofthe first, second and third blister actuators in their second positionsfor at least 2 seconds, preferably at least 5 seconds, preferably atleast 8 seconds, preferably at least 10 seconds to collapse the blistersand eject the reagents contained therein.

Preferably each blister actuator has a travel of between 20 mm and 70 mmbetween its first and second positions, preferably between 40 mm and 50mm, preferably 44 mm.

Preferably, one or more of the first, second and third blister actuatorscomprises a spring mounted around the stem portion, between the housingportion and the tip portion to provide damping.

The invention also provides a fluidic cartridge for carrying out adiagnostic test on a sample contained therein, the cartridge comprisingfirst, second and third collapsible blisters containing at least onereagent for use in the diagnostic test and a mechanical valve forisolating the sample with the cartridge, and configured for use with acartridge reader of any one of claims 2 to 8, the first, second andthird collapsible blisters configured to be collapsed by the first,second and third blister actuators, respectively, and the mechanicalvalve configured to be moved from an open position to a closed positionby the mechanical valve actuation assembly.

In a second aspect, the present invention provides a cartridge readerfor carrying out a diagnostic test on a fluid sample contained in afluidic cartridge in the cartridge reader, the fluidic cartridgecomprising bellows for moving the fluid sample along a network ofchannels, a plurality of pneumatically actuated valves in the network ofchannels and a pneumatics interface, the cartridge reader comprising: apneumatics system, comprising:

a pneumatics interface, comprising a plurality of pneumatic interfaceports, configured to couple to the pneumatics interface on the fluidiccartridge;

a positive pressure sub-system comprising first and second positivepressure reservoirs and a first pump configured to provide a supply ofpositive pressure to maintain the first and second positive pressurereservoirs at first and second positive pressures, respectively, whereinthe first and second positive pressures are different; and

a negative pressure sub-system comprising a negative pressure reservoirand a second pump configured to provide a supply of negative pressure tomaintain the negative pressure reservoir at a negative pressure;

a valve system comprising a plurality of solenoid valves, each connectedto a pneumatic interface port and configured to couple to that pneumaticinterface port at least one of: the first and second positive pressurereservoirs and the negative pressure reservoir; and processing meansconfigured to operate the first and second pumps and the plurality ofsolenoid valves according to a predetermined cycle during the diagnostictest such that during the cycle: a first subset of pneumatic interfaceports is selectively coupled to either the first positive pressurereservoir or the negative pressure reservoir for actuating the bellowsand the pneumatic valves on the cartridge; and

a second subset of pneumatic interface ports is coupled to the secondpositive pressure reservoir for evacuating a surplus fluid from at leasta portion of the network of channels in the fluidic cartridge.

Preferably, the positive pressure sub-system further comprises apressure regulator connected between the second positive pressurereservoir and a reference-pressure solenoid valve, and wherein theprocessing means is configured to operate the reference-pressuresolenoid valve such that a reference-pressure pneumatic interface portis coupled to the second positive pressure reservoir via the pressureregulator for providing a reference pressure to the fluidic cartridge.

Preferably, the first positive pressure is a positive gauge pressure of1 bar.

Preferably, the negative pressure is a negative gauge pressure of 0.5bar.

Preferably, the reference pressure is a gauge pressure of between 0 and1 bar and the pressure regulator is configured to maintain the referencepressure at 0.5% full scale accuracy.

Preferably, the positive pressure sub-system further comprises one ormore fluid traps between a corresponding one or more pneumatic interfaceports and their respective solenoid valves.

Preferably the one or more fluid traps includes a fluid trap betweeneach of the second subset of pneumatic interface ports and theirrespective solenoid valves.

Preferably, the one or more fluid traps includes a fluid trap betweenthe reference-pressure solenoid valve and the reference-pressurepneumatic interface port.

Preferably the pneumatic interface comprises eleven pneumatic interfaceports aligned in first and second rows, consisting of five pneumaticinterface ports and four pneumatic interface ports, respectively,wherein the pneumatic interface ports of the first and second rows areoffset from each other.

Preferably pneumatic interface port number 1 is situated at one end ofthe first row, and each subsequently numbered port is adjacent thepreceding port of the opposite row.

Preferably the first subset of pneumatic interface ports consists ofport numbers 1, 2, 3, 4, 6, 8, 10 and 11.

Preferably the second subset of pneumatic interface ports consists ofport numbers 5 and 7.

Preferably the reference-pressure pneumatic interface port is portnumber 9.

Preferably the positive pressure sub-system, negative pressuresub-system and valve system and provided on a pneumatics block, and thepneumatics interface ports are provided on a pneumatics interfacemanifold that is connected to the pneumatics block by a plurality ofpneumatics pipes.

Preferably the reader comprises a fixed upper clamp and a movable lowerclamp configured to hold a cartridge within the cartridge reader, andwherein the pneumatics interface manifold is mounted to the upper clampby one or more springs.

Preferably the one or more springs are configured to apply a biasingforce of between 30 and 60N, preferably between 40 and 50N, preferably45N.

The invention also provides a fluidic cartridge for carrying out adiagnostic test on a fluid sample contained therein, the cartridgecomprising bellows for moving the fluid sample along a network ofchannels, a plurality of pneumatically actuated valves in the network ofchannels and a pneumatics interface, and configured for use with acartridge reader of any one of claims 10 to 24, the pneumatics interfaceon the cartridge configured to couple with the pneumatics interface onthe reader such that the reader is capable of actuating the bellows andthe pneumatic valves on the cartridge, and evacuating a surplus fluidfrom at least a portion of the network of channels in the fluidiccartridge.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of an exemplary fluidic cartridge in whichthe invention may be provided.

FIG. 2 is a top view of an exemplary fluidic cartridge in which theinvention may be provided.

FIG. 3 is an exploded view of the exemplary fluidic cartridge of FIG. 2.

FIG. 4 is a perspective view of the housing of the exemplary fluidiccartridge of FIG. 2.

FIG. 5 is a perspective view of the blister sub-assembly of theexemplary fluidic cartridge of FIG. 2.

FIG. 6A is a top view of the pneumatic layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 6B is a bottom view of the pneumatic layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 7 is a top view of the pneumatic foil of the exemplary fluidiccartridge of FIG. 2.

FIG. 8A is a top view of the fluidic layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 8B is a bottom view of the fluidic layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 9 is a top view of the fluidic foil of the exemplary fluidiccartridge of FIG. 2.

FIG. 10 is a top view of the electrode layer of the exemplary fluidiccartridge of FIG. 2.

FIGS. 11a and 11b shows a front view of exemplary reader.

FIGS. 12a and 12b show an exploded view of the housing of the readershown in FIGS. 11a and 11 b.

FIG. 13 shows a bottom view of a base of the reader shown in FIGS. 11aand 11 b.

FIG. 14 shows a schematic view of the principal sub-systems comprised inan exemplary reader.

FIG. 15 shows a schematic view of the internal components of theexemplary reader organised onto six boards.

FIG. 16 shows a cross section of the reader of FIGS. 11a and 11 b.

FIGS. 17a and 17b show a drawer of the exemplary reader.

Figured 18 a and 18 b show the drawer of FIGS. 17a and 17b slidablymounted in an upper clamp of the reader.

FIG. 19 shows a drawer latching mechanism of the reader.

FIG. 20 shows a cross section of an exemplary cartridge inserted intothe reader.

FIGS. 21a and 21b shows a plan view of the upper clamp and the lowerclamp respectively.

FIG. 22 shows a cross section of a clamping assembly of the exemplaryreader.

FIG. 23a , FIG. 23b and FIG. 23c each shows a schematic of the stepsperformed by the reader during a test cycle.

FIG. 24 shows an isolation valve latching mechanism comprised in theupper clamp.

FIG. 25 is a plan view showing first, second and third thermal stacksarranged on the lower clamp of the exemplary reader.

FIGS. 26a-c show a thermal stack comprised in the exemplary reader.

FIG. 27 shows an aluminium spreader plate provided in the thermal stackof FIG. 26.

FIG. 28 shows a plan view of the exemplary cartridge with first, secondand third zones to heated and/or cooled by the thermal stacks in FIG.25.

FIG. 29 is a plan view of the upper clamp, comprising first, second andthird blister actuators and a mechanical valve actuator.

FIG. 30 shows the tip geometries of the first, second and third blisteractuators from FIG. 29.

FIG. 31a shows the tip geometry of the mechanical actuator from FIG. 29.

FIG. 31b shows the valve cavity of a mechanical valve comprised in anexemplary cartridge.

FIG. 32 is a perspective view of a pneumatics block comprised in thereader.

FIG. 33 is a top view of the pneumatics block of FIG. 32.

FIG. 34 is a side view of the pneumatics block of FIG. 32

FIG. 35 shows an interface on the manifold to which the pneumatic pipesfrom the pneumatic block are connected.

FIG. 36 shows an interface on the manifold which is configured to coupleto a corresponding interface on the cartridge.

FIG. 37 shows the pneumatic interface located on the upper clamp.

FIG. 38a , FIG. 38b , and FIG. 38c each shows a pneumatic circuitdiagram for the pneumatic block.

FIG. 39 shows a portion of the housing of the exemplary cartridge inwhich the invention is implemented.

FIG. 40 shows an exemplary circuit containing the electrodes.

FIG. 41 shows the results of a test using differential pulse voltammetryor square wave pulse voltammetry.

FIG. 42 shows the forward and reverse current for ferrocene in a buffer.

FIG. 43 is a section view of an exemplary valve.

FIG. 44 is a section view of another exemplary valve in in an openposition.

FIG. 45 is a section view of the valve of FIG. 44 in an intermediateposition.

FIG. 46 is a section view of the valve of FIG. 44 in a closed position.

FIG. 47 is a schematic diagram of an exemplary valve system.

DETAILED DESCRIPTION

Embodiments of the invention will now be described in the context of anexemplary cartridge reader for reading an exemplary cartridge. Whilstnot necessary to understand the present invention, it is beneficial toprovide a general description of the principles of the structure,manufacture, function and use of the cartridge and associated methods ofperforming a test.

The exemplary cartridge and cartridge reader and associated methodschosen to illustrate the present invention are for the detection of apathogenic bacterium using a nucleic acid extraction process, followedby PCR amplification and electrochemical detection. However, the skilledperson would understand that the invention is not limited to theexemplary reader and associated methods, and is suitable for use in awide variety of sample analysis techniques or biological assays; forexample, assays of any target nucleic acid sequences in a liquid sample.

Those skilled in the art will understand that the devices and methods ofthe invention described herein and illustrated in the accompanyingdrawings are non-limiting exemplary embodiments and that the scope ofthe present invention is defined solely by the claims. The featuresillustrated or described in connection with one exemplary embodiment maybe combined with the features of other embodiments. Such modificationsand variations are included within the scope of the present disclosure.

The exemplary cartridge reader is intended for use with an exemplaryfluidic cartridge, which comprises: a fluidic portion through which thesample flows and in which nucleic acid extraction, amplification anddetection take place; a pneumatic portion which controls flow throughthe fluidic portion; a sample processing region for performing nucleicacid on the sample, PCR and at least two electrodes which provide apotential difference for the detection of an amplified nucleic acid ofinterest The fluidic portion and pneumatic portion may be constructed ofa fluidic layer, a fluidic foil, a pneumatic layer and a pneumatic foilsuch as those described in relation to the exemplary cartridge below.However, the fluidic portion does not necessarily consist only of afluidic layer and a fluidic foil and the pneumatic portion does notnecessarily consist only of a pneumatic layer and a pneumatic foil.Rather, the layers may interact to produce the fluidic portion and thepneumatic portion such that parts of all or some of the layers make upeach portion. Rather than referring to the particular layers of thecartridge, the fluidic portion refers to the particular areas of thecartridge which provide the function of allowing controlled sample flow,and the pneumatic portion refers to the particular areas of thecartridge which provide the function of controlling the flow through thefluidic portion.

The housing, fluidic portion and pneumatic portion are made of plastic.By plastic is meant a synthetic or natural organic material that may beshaped when soft and then hardened, including resins, resinoids,polymers, cellulose derivatives, casein materials, and protein plastics.Examples of plastics from which the cartridge may be constructedinclude, but are not limited to thermoplastics, for examplepolycarbonate, polyethylene terephthalate, cyclic olefin copolymers suchas Topaz, acrylonitrile butadiene styrene, and thermoplastic elastomers,for example polypropylene. Plastic housings, fluidic portions andpneumatic portions can include components which are not made of plastic(e.g. blisters made from metal foil, metallic inserts at the sampleinlet), but they are formed primarily from plastic. The use of plasticmaterials facilitates economical manufacture of the cartridges.

Whilst the pneumatic and fluidic foils may be made from a metal foil,the preferred materials are plastic including those mentioned above. Inparticular, it is preferred that foils are a polyethyleneterephthalate/polypropylene composite, with the polypropylene in contactwith the fluidic sample.

The target nucleic acid sequence is any nucleic acid to be detected in asample. The target nucleic acid(s) to be amplified and detected in thecartridge will usually be DNA, but it is also possible to amplify anddetect RNA. In some embodiments a cartridge may permit amplificationand/or detection of both DNA and RNA targets.

The liquid sample is the composition which is introduced into thecartridge in order to determine whether the target nucleic acid(s) ofinterest is/are present. The sample may be a composition in which thenucleic acid to be detected is suspected to be present (e.g. forclinical diagnosis), or may be a composition in which the nucleic acidto be detected is potentially present (e.g. for contamination testing).

The liquid sample can have various sources. For instance, it can bematerial obtained from an animal or plant (e.g. for diagnosis ofinfections or for genotyping). Such samples may be obtained with minimalinvasiveness or non-invasively, e.g., the sample may be obtained from ananimal using a swab, or may be a bodily fluid. As an alternative, thesample may be material obtained from food or water (e.g. forcontamination testing). The sample will usually include cells, and thetarget nucleic acid (if present) can be extracted from these cellswithin the cartridge. One skilled in the art will appreciate thatsamples can be diluted or otherwise treated prior to being introducedinto the cartridge, but it is preferred that the cartridge can handlematerial which has not been pre-treated in this way.

An animal from whom the sample is obtained may be a vertebrate ornon-vertebrate animal. Vertebrate animals may be mammals. Examples ofmammals include but are not limited to mouse, rat, pig, dog, cat,rabbit, primates or the like. The animal may be a primate, and ispreferably a human. Thus the cartridge can be used for clinicaldiagnosis of human samples.

In addition to analysing a sample, the cartridge can analyse a positiveand/or negative control to provide confirmation that the cartridge isfunctioning as expected. The control(s) can be introduced into thecartridge by a user, or can be included within a cartridge before use.

The inclusion of a positive internal control nucleic acid allows a userto identify whether a negative result for the sample has been obtainedbecause the nucleic acid amplification has been unsuccessful (invalidresult). If the positive control nucleic acid fails to be detected inthe detection chamber, despite its presence in an amplification chamber,the user will be able to identify the test as an invalid result, and canperform another test.

A positive control nucleic acid may be any nucleic acid that will not befound in a sample used in the cartridge. The internal control DNA may betaken from a bacterium that is not pathogenic to animals and whichcontains a nucleic acid that is highly specific to the bacterium. Oneexample of a possible bacterium from which the control nucleic acid maybe taken for an animal sample is Pectobacterium atrosepticum, althoughany control nucleic acid may be used that will not be present in asample.

The fluidic portion of the cartridge comprises channels and chambersthrough which sample flows. The flow of sample through the cartridge iscontrolled in two ways. Firstly, the fluidic portion has a gas inlet.The gas inlet is connected to a gas supply, and injection of gas intothe fluidic portion via this inlet allows the sample to be pusheddownstream through the cartridge, towards the detection chamber. The gassupply may be provided by the reader. As an alternative, the gas supplymay be an on-board gas supply. Preferably, the gas supply is provided byan external source and the gas inlet is connected to a pneumatic circuitsuch that the gas supply is provided via a pneumatic inlet on thecartridge. Secondly, at least one pneumatically controlled valvecontrols local movement of the sample through the fluidic portion. Thepneumatically controlled valve(s) may be controlled independently ofother pneumatically controlled valves and may be controlledindependently of the gas supply that generally causes downstreammovement of the sample via the gas inlet. The gas inlet and thepneumatically controlled valve(s) also permit sample to be flushedthrough the fluidic portion e.g. to exclude excess volumes of material.The fluidic portion also has an exhaust which allows air and wastematerial to exit the channels and chambers of the fluidic portionwithout a build-up of pressure occurring in the cartridge. Preferably,the exhaust comprises a waste chamber and/or a waste vent.

The fluidic portion of the cartridge includes reagents and/or physicalcomponents for cell lysis and nucleic acid separation. These may be anyreagents or physical components that are capable of lysing cells andseparating nucleic acids from cell debris and other cellular components.For instance, they may comprise (i) a lysis buffer which is capable ofcausing lysis of target cells which may be present in the sample e.g.buffers including a detergent such as nonyl phenoxypolyethoxylethanol(available as NP-40) or toctylphenoxypolyethoxyethanol, (available asTriton X 100), or including guanidine thiocyanate, and/or (ii) a capturesupport or column which specifically binds nucleic acids but does notbind other undesired cellular components (e.g. proteins and lipids). Thecapture column comprises a capture filter and may additionally comprisea depth filter. The filters may be made of glass fibres (available asWhatman filters), or may be made of silica, although any column orsupport which is capable of separating nucleic acids from other cellularcomponents may be used. Elution using a wash buffer to remove celldebris and other cellular components, followed by elution using anelution buffer to elute the separated nucleic acids from the capturesupport or column can be undertaken such that the capture column canseparate nucleic acids from cell debris and other cellular components.

A channel through which the sample flows fluidly connects the sampleinlet to at least one amplification chamber where nucleic acidamplification can take place. The purpose of the amplificationchamber(s) is to permit amplification of any target nucleic acid ofinterest that is present in the sample (and, where present, any positivecontrol nucleic acid). Any nucleic acid amplification method may be usedand these are described in more detail below in relation to an exemplarycartridge. The different nucleic acid amplification reagents that arerequired for different nucleic acid amplification methods are well knownin the art. These reagents are provided in or upstream of theamplification chamber(s) such that the sample (and any positive control)includes all necessary reagents for nucleic acid amplification once itreaches the amplification chamber. Adaptation of a nucleic acidamplification method according to the target nucleic acid to be detectedis also well known in the art (e.g. design of primers). The skilledperson would therefore be able to adapt the reagents for nucleic acidamplification accordingly. The term “chamber” does not denote anyparticular size or geometry, but instead it means a region within thefluidic portion which is designed to permit nucleic acid amplificationto occur. Thus, for instance, it could be a region in which the samplecan be fluidically isolated (e.g. via the use of pneumaticallycontrolled valves) while the steps required for nucleic acidamplification (e.g. thermocycling, etc.) occur, and it can be locatedwithin the cartridge so that it is in the proximity of any externalresources that are needed (e.g. next to a heat source within a cartridgereader, thereby permitting thermal cycling to occur).

Multiple test amplification channels and/or chambers may be included inthe cartridge. The different test amplification channels and/or chambersmay include reagents required to amplify different nucleic acids ofinterest Therefore using multiple amplification test channels and/orchambers allows multiple tests to be performed on a single cartridge,simultaneously (including any controls). As an alternative, reagents foramplification of multiple different nucleic acids may be present in asingle amplification chamber, and the different nucleic acids (whethermultiple target nucleic acids, or a target nucleic acid and a controlnucleic acid) may be amplified simultaneously in the same amplificationchamber.

A further channel through which the sample flows after nucleic acidamplification fluidly connects the at least one amplification chamber toat least one detection chamber where the results of nucleic acidamplification can be detected. In or upstream of the detection chamberare reagents for nucleic acid detection such that the sample includesall necessary reagents for the detection once it reaches the detectionchamber. The reagents for nucleic acid detection may be specific for theparticular target nucleic acid, i.e. they may allow for detection of thepresence of the specific nucleic acid sequence. As an alternative, thereagents for nucleic acid detection may be generic reagents to detectthe presence of any nucleic acids. Such generic reagents may be used ifall nucleic acids other than the target nucleic acid are removed priorto detection. For example, this may be achieved by providing a nucleasethat is capable of hydrolysing all nucleic acids present in the sampleother than the target nucleic. The amplified target nucleic acid can beprotected from hydrolysis, for example by inclusion of chemicalmodifications in the primers which are incorporated into the amplifiedproduct and which cannot be hydrolysed. Reagents for nucleic aciddetection are described below in relation to an exemplary cartridge butusually comprise a probe including an electrochemical label. The probeis capable of hybridising to the amplified nucleic acid which has beenamplified in the amplification chamber(s). Following hybridisation ofthe probe to the amplified nucleic acid, the detection of the nucleicacid may occur via a detectable change in the signal from the label. Inpractice, this involves three stages: binding of a probe to the targetDNA, cleaving of the label from the probe by T7, followed by detectionof the signal from the label. In some embodiments the change may becaused by hydrolysis of the probe. Where the probe is hydrolysed,hydrolysis is usually achieved using a double strand specific nuclease,which can be an exonuclease or an endonuclease. Preferably, the nucleaseis T7 endonuclease. The signal from the label is capable of undergoing achange following hydrolysis of the probe. This is due to a change in theenvironment of the label when it moves from being bound to the rest ofthe probe to being free from the rest of the probe or bound to a singlenucleotide or a short part of the probe. Further details of the types ofprobes and detection methods that may be used can be found in Hillier etal. Bioelectrochemistry, 63 (2004), 307-310. As an alternative, methodsfor causing a detectable change in the signal from the label which donot rely on hydrolysis of the probe may be used e.g. see Ihara et al.Nucleic Acids Research, 1996, Vol. 24, No. 21 4273-4280. This change inenvironment of the label leads to a change in the signal from the label.The change in signal from the label can be detected in order to detectthe presence of the nucleic acid of interest.

Where a positive control nucleic acid is used, the reagents for nucleicacid detection will additionally include a positive control probeincluding a label. The positive control probe is capable of hybridisingto the amplified control nucleic acid. The signal provided by the labelsof the positive control and target probes may be the same, but presentin separate detection chambers such that the signals corresponding tothe control and test nucleic acids can be distinguished. As analternative, the signal provided by the labels of the control and targetprobes may oxidise at different voltages, such that the signals aredistinguishable from one another, even if the probes are present in thesame detection chamber.

Multiple test detection channels and/or chambers may be included in thecartridge. The different test detection channels and/or chambers mayinclude reagents required to detect different nucleic acids of interest.Therefore using multiple detection test channels and/or chambers allowsmultiple tests to be performed on a single cartridge, simultaneously. Asan alternative, reagents for detection of multiple different nucleicacids may be present in a single detection chamber, and the differentnucleic acids (whether multiple target nucleic acids or a target nucleicacid and a control nucleic acid) may be detected simultaneously in thesame detection chamber.

The label is detectable by use of the cartridge's electrodes, and so thelabel will usually be an electrochemical label, such as a ferrocene.Examples of labels which may be used can be found in WO03/074731,WO2012/085591 and PCT/GB2013/051643. Signal emitted by the label can bedetected by a cartridge reader.

The pneumatic portion of the cartridge comprises at least one pneumaticcircuit which each control at least one pneumatically controlled valve.The pneumatic portion controls sample flow through the cartridge by theopening and closing of pneumatically controlled valves. The opening andclosing of the valves is controlled by changes in pneumatic pressure inthe pneumatic circuit that is applied through a pneumatic pressureinlet. Usually, the cartridge contains many pneumatically controlledvalves. The pneumatically controlled valves may be controlled byseparate pneumatic pressure inlets. These valves can be used to preventdownstream movement of sample through the fluidic portion untilnecessary steps have been performed and/or to prevent unwanted reversemovement of sample upstream. For example, a valve may be providedupstream of the at least one amplification chamber in order to preventdownstream movement into the at least one amplification chamber untilcell lysis and nucleic acid separation has taken place. Following celllysis and nucleic acid separation the valve upstream of the at least oneamplification chamber may be opened in order to allow downstream flow.It can then be closed again, to prevent backflow out of the chamber backtowards the sample inlet.

The cartridge comprises at least two electrodes which can provide apotential difference across the at least one detection chamber. Thepotential difference causes current to flow through the at least onedetection chamber, thereby permitting the detection of signal fromelectrochemically active labels.

The cartridge reader generally comprises a cartridge receiving regioninto which a cartridge containing a sample may be inserted; first andsecond clamps, between which the cartridge is held during a test cycle;a pneumatics assembly, for coupling to pneumatic ports on the exemplarycartridge and actuating one or more pneumatic valves comprised withinthe exemplar cartridge; a thermal module, which comprises one or morethermal stacks for heating various sample handling zones in the fluidiccartridge, an electronics interface, configured to couple to the atleast two electrodes and receive an electrical signal therefrom; and acontrol unit comprising the processing means required for controllingthe test and reading a result therefrom. The exemplary cartridge readermay also comprise: one or more mechanical actuators for actuating one ormore collapsible blisters provided on the fluidic cartridge andconfigured to eject a fluid contained therein into the network ofchannels in the fluidic device; a mechanical valve actuator foractuating a mechanical valve comprised within the cartridge; anisolation actuator, configured to actuate an isolation valve comprisedon the fluidic cartridge in the event of a fault, power loss or at theend of a test. The cartridge reader may also comprise means foridentifying a cartridge inserted into the reader, such as, for example,a bar code reader for reading information from a bar code label affixedto the cartridge.

1. The Exemplary Cartridge 1.1 Overview

The exemplary cartridge described below is intended to be a single-use,disposable cartridge for performing a test on a sample introduced intothe cartridge. The exemplary cartridge is a fluidic cartridge withchannels of an appropriate scale (as detailed hereafter). However, theinvention may be performed on a microfluidic device, or an LOC. Once thetest has been run, it is preferred that the cartridge is disposed of.However, if desired, the cartridge may be sent for re-processing toenable it to be used again.

It is preferred that the cartridge comprises all of the biologicalagents necessary for conducting the test of choice. For example, theexemplary cartridge is used for detecting the presence, absence oramount of a pathogen of interest. Any pathogen may be detected. Examplesof pathogens which may be detected by the cartridge are Chlamydiatrachomatis, Trichomonas vaginalis, Neisseria gonorrhoea, Mycoplasmagenitalium and methicillin resistant Staphylococcus aureus. To that endthe cartridge comprises buffers for lysis the bacteria, washing thedebris to waste and a clean buffer for re-suspending the target DNA. Thecartridge also comprises dry reagents for nucleic acid amplification.Nucleic acid amplification may be performed using any nucleic acidamplification method. The nucleic acid amplification method may be athermocycling method in which the temperature at which the method isperformed is varied such that different steps of the amplification areable to take place at different temperatures within the cycle. Forexample melting, annealing of primers and extension may each beperformed at different temperatures. By cycling through thetemperatures, the timing of each of the steps of the method can becontrolled. As an alternative, the nucleic acid amplification may be anisothermal method in which the temperature is kept constant. In both thethermocycling and the isothermal nucleic acid amplification methods, thetemperature is controlled during nucleic acid amplification.

Examples of nucleic acid amplification methods are the polymerase chainreaction (PCR), the ligase chain reaction (LCR), strand displacementamplification (SDA), transcription mediated amplification, nucleic acidsequence-based amplification (NASBA), helicasedependent amplificationand loop-mediated isothermal amplification. The reagents for nucleicacid amplification will vary depending of the nucleic acid amplificationmethod used but include a polymerase and nucleotide triphosphates.

As explained below, the cartridge also comprises detection reagentswhich are capable of detecting the presence or absence of amplifiednucleic acids which are the product of the nucleic acid amplificationmethod. The reagents for nucleic acid detection comprise a probe whichis capable of hybridising to the amplified nucleic acid. The probeincludes a ferrocene label. Following hybridisation of the probe to theamplified nucleic acid, the detection of the nucleic acid occurs via adetectable change in the signal from the label. The change is caused byhydrolysis of the probe, which is achieved using a double strandspecific nuclease. The nuclease is a T7 endonuclease. The ferrocenegives different electrochemical signals when it is part of a probe orwhen it is attached only to a single nucleotide, and so hydrolysis iseasily detected. Thus, the change in signal from the label permitsdetection of the presence of the nucleic acid of interest.

The electrodes allow the detectable change in the signal from the label,which occurs in the presence of the target nucleic acid, to be detected

The cartridge is configured for use with a cartridge reader (not shown).The cartridge comprises a number of pneumatic, mechanical, thermal andelectrical interfaces (described in more detail below) through which thereader interacts with the cartridge to perform the test. Hence, in use,the cartridge would be inserted into the reader, and the reader would beactivated to begin interacting with the cartridge via the interfaces toperform the test. For the purposes of understanding the presentinvention, it is not necessary to describe exactly how the cartridgeinteracts with the reader to conduct a particular test and provide thetest results, but an overview of an exemplary operation of a cartridgeis provided hereafter.

1.2 Schematic Diagram of the Exemplary Cartridge

Before explaining the structure and arrangement of the components of anexemplary fluid cartridge in detail, it is helpful to describe thelayout of the exemplary cartridge at a high level with reference to theschematic shown in FIG. 1.

It is convenient to consider the overall layout of the cartridge interms of the flow of liquids, including the liquid sample, through thecartridge. Unless otherwise specified hereafter, the passage of liquidsincluding the liquid sample and the liquid buffers is referred to as the‘fluid pathway’ which has an upstream end and a downstream end. Unlessotherwise specified hereafter, ‘downstream’ generally refers to thedirection of flow of the liquids and ‘upstream’ refers to the directionopposite the direction of flow. The fluid pathway in the exemplarycartridge may have different branches (and thus form different fluidpathways), but all pathways have a recognisable direction of flow whichpermit a skilled person to identify the upstream and downstreamdirections. However, there is an exception to this general definition,which is when the liquid sample is pumped between the mixing chamber 10and the bellows 20. In this case, fluid is intermittently pumped backupstream in the opposite direction to its general direction of fluidflow, which is downstream. This mixing serves to mix the lysis andsample and to rehydrate the internal control.

The liquid sample is introduced into the cartridge at a sample mixingchamber 10 through an entry port. A particular arrangement of apreferred entry port may itself form an isolated inventive aspect of thecartridge, as described further in section 3, below. A sample indicator12 is fluidly coupled to the sample mixing chamber 10 such that a sampleintroduced into the sample mixing chamber 10 is visible in the sampleindicator 12. Also connected to the sample mixing chamber 10 is ablister 14 containing a lysis buffer. The lysis buffer comprisesguanidine thiocyanate. Once the sample has been introduced into thesample mixing chamber 10, and a test is started, the lysis blister 14 iscollapsed so as to expel the lysis buffer into the sample mixing chamber10 where it mixes with the liquid sample introduced therein.

Downstream of the sample mixing chamber 10, along a main channel 16, isa coarse filter 18. The coarse filter 18 filters out any large debris inthe liquid sample, such as skin or bodily hair, as the liquid samplepasses through main channel 16.

Downstream of the coarse filter 18, along the main channel 16, is abellows 20 having an upstream bellows valve 22 a and a downstreambellows valve 22 b. As described in more detail below, the bellows 20,together with its upstream and downstream valves 22 a-b, is capable ofpumping the liquid sample from the upstream end of the fluid pathway(i.e. from the sample mixing chamber 10) to the downstream end. Insummary, this is achieved by virtue of flexible membranes within thebellows 20 and the upstream and downstream bellows valves 22 a-b whichactuate to create local pressure differentials to, on the one hand, drawin the liquid sample from the sample mixing chamber 10 into the bellows20 and, on the other hand, from the bellows 20 further downstreamthrough the main channel 16. This is achieved by carefully choreographedpneumatic actuation of the flexible membranes in the valves. Particulararrangements of a preferred valve may themselves form isolated inventiveaspects of the cartridge, as described further in section 3, below.

Downstream of the bellows along the main channel 16 is a capture column24. The purpose of the capture column 24 is to separate nucleic acidsfrom cell debris and other cellular components. The capture columncomprises a capture filter and a depth filter both made of glass fibres.A particular arrangement of a preferred capture column may itself forman isolated inventive aspect of the cartridge, as described further insection 3, below.

Two branch channels 26, 28 join the main channel 16 between thedownstream bellows valve 22 b and the capture column 24. The purpose ofthe branch channels is to introduce liquid buffers necessary forperforming the desired test. For example, with the test conducted by theexemplary cartridge, it is necessary to introduce an elution buffer anda wash buffer into the main channel once the sample has passed through.The wash buffer is contained in a wash buffer blister 30 and the elutionbuffer is contained in an elution buffer blister 32. The introduction ofthe wash buffer and elution buffer into the main channel 16 iscontrolled by wash buffer valve 34 and elution buffer valve 36,respectively. At the appropriate point in the test, the wash and elutionbuffer blisters 30, 32 are collapsed so as to expel the wash and elutionbuffers into the branch channels 26, 28 and thence into the main channel16 through the wash and elution buffer valves 34, 36.

Downstream of the capture column 24, along a waste branch 16 a of themain channel 16, is a waste chamber 38. A particular arrangement of apreferred waste chamber may itself form an isolated inventive aspect ofthe cartridge, as described further in section 3, below. The purpose ofthe waste chamber 38 is to collect the cell debris and cellularcomponents other than nucleic acids and contain them, thereby preventingthem from entering the test channel 54 a or the control channel 54 b.The waste chamber 38 is vented to atmosphere through a waste vent 40,and an aerosol impactor 42 is provided between the waste chamber 38 andthe waste vent 40 to prevent particulate matter from escaping from thewaste chamber 38 into the atmosphere. A waste chamber valve 44 in themain channel waste branch 16 a of the main channel 16 permits andprevents fluids passing into the waste chamber 38 at appropriate pointsduring the test.

Downstream of the capture column 24, along an elution branch 16 b of themain channel 16, is an elution chamber 46. The purpose of the elutionchamber 46 is to allow the sample preparation to settle and for bubblesto disperse before the sample enters the amplification chambers. Anelution chamber valve 48 in the elution branch 16 b of the main channel16 permits and prevents fluids passing into the elution chamber 46 atappropriate points during the test.

Downstream of the elution chamber 46 is a convoluted mixing channel 52.H ere the prepared sample is mixed prior to passing through theisolation valve 50.

In the present application, the components upstream of the isolationvalve 50 are referred to as being comprised in the ‘front end’ of thecartridge, whilst the components downstream of the isolation valve 50are referred to as being comprised in the ‘back end’ of the cartridge.Broadly speaking, the liquid sample is prepared for analysing in thefront end of the cartridge, and the analysis is carried out on thesample in the back end of the cartridge.

The isolation valve 50 is open to permit the prepared liquid sample topass from the front end to the back end of the cartridge. At anappropriate point in the test, after the liquid sample has been preparedand is within the back end of the cartridge for analysis, the isolationvalve 50 is closed to prevent any of the sample from re-entering thefront end. Once the isolation valve 50 is closed, it cannot be openedagain. The isolation valve 50 also acts as a safeguard in case of apower failure, wherein the reader closes the isolation valve 50 toprevent leakage.

Downstream of the isolation valve 50, the fluid pathway splits into anamplification test channel 54 a and an amplification control channel 54b. Each of the amplification channels 54 a-b comprises an amplificationchamber 56 a-b having an amplification chamber inlet valve 58 a-b and anamplification chamber outlet valve 60 a-b. Any nucleic acidamplification method may be performed in the nucleic acid amplificationchamber. If PCR is used, the nucleic acid amplification chambers containa thermostable DNA polymerase, dNTPs, a pair of primers which arecapable of hybridising to the nucleic acid to be amplified. Optionally,the nucleic acid amplification chambers may additionally contain buffersalts, MgCl₂, passivation agents, uracil N-glycosylase and dUTP. Anexample of a thermostable DNA polymerase that may be used is Taqpolymerase from Thermus aquaticus.

Each of the nucleic acid amplification chambers in the exemplarycartridge comprises reagent containment features in the form of firstand second shallow wells formed in the fluidic layer. The reagents to beused in the cartridge are spotted in the wells. In the exemplarycartridge, the test-specific reagents and the generic reagents areisolated from each other by spotting each in a different well. Hence,the test-specific reagents are spotted in a first well in the chamberand the generic reagents are spotted in a second well in the chamber. Byspotting the reagents separately, it is easier to swap the test-specificreagents during manufacture for a different set of test-specificreagents, so as to perform a different test, whilst keeping the genericreagents as they are.

In the exemplary cartridge, the ratio of nucleic acid amplificationchambers to detection chambers is 1:2. The prepared sample enters theback end of the cartridge at the isolation valve 50 and is split intotwo nucleic acid amplification chambers. After processing, the each ofthe two processed measures of sample from the nucleic acid amplificationchamber is split into two detection chambers. Therefore, for each sampleintroduced into the exemplary cartridge, four detection chambers may befilled from two nucleic acid amplification chambers, thus facilitatingduplex amplification and 4-plex detection.

However, it will be appreciated that one or three or more nucleic acidamplification chambers may be provided to provide any level ofmultiplexing desired, and that the number of the detection chambersprovided may be adjusted accordingly to maintain a 1:2 ratio of nucleicacid amplification chambers to detection chambers.

The ratio 1:2 is preferred for the exemplary cartridge because such aratio allows twice the number of target nucleic acids to be assayedcompared to the number of different labels required for detection in thedetection chambers. However, it will be appreciated that the ratio maybe changed depending on the number of labels and PCR targets for theliquid sample. For instance, the ratio may be 1:1, 1:3 or 1:n such thatthere are n detection chambers branching from the main channel of eachfluid pathway when there are n times as many multiplexed PCR targets forthe number of labels.

PCR primers specific for Chlamydia trachomatis are dried down in theamplification chamber in the amplification test channel together withthe other reagents required for nucleic acid amplification. PCR primersspecific for a positive control nucleic acid are dried down in theamplification chamber in the amplification control channel together withthe other reagents required for nucleic acid amplification. A positivecontrol nucleic acid is also provided in the amplification chamber inthe amplification control channel, taken from Pectobacteriumatrosepticum. The dried down reagents are reconstituted when the liquidsample reaches them.

Downstream of the amplification chamber outlet valves 60 a-b each of theamplification channels 54 a-b splits into two further detectionchannels, leading to two detection chambers for each amplificationchamber, giving a total of four detection chambers 62 a-d in total. Thereagents for nucleic acid detection, including the target probe, aredried down in the detection chambers 62 a-d downstream of the testamplification chamber 56 a or 56 b. The reagents for nucleic aciddetection including the control probe are dried down in the detectionchambers downstream of the control amplification chamber 56 a or 56 b(whichever is not the test chamber mentioned above). Each detectionchamber 62 a-d is provided with its own gas spring 64 a-d which forms adead end at the downstream end of the fluid pathway.

Reagents for nucleic acid detection are provided in detection chambers.The reagents for nucleic acid detection include probes having aferrocene label. These probes are capable of hybridising to theamplified nucleic acids. Following hybridisation of the probes to theamplified nucleic acids, the probes are hydrolysed by a double strandspecific nuclease which causes the label to be freed from the rest ofthe probe. As explained above, freeing of the label from the rest of theprobe causes a detectable change in the signal from the label. Thecontrol probe is provided in separate detection chambers to the targetprobe and detection of the target nucleic acid and the control nucleicacid take place in different detection chambers, such that the signalsare distinguishable from one another.

Downstream of the amplification outlet valves 60 a-b, but upstream ofthe forks creating the four detection channels, two bypass channels 66a-b respectively join the two amplification channels 54 a-b. The purposeof the bypass channels 66 a-b is to remove excess liquid sample withinthe amplification channels 54 a-b before the liquid sample enters thedetection chambers 62 a-d. The bypass channels 66 a-b connect to abypass valve 68, which is also fluidly coupled to the elution chamberbranch 16 b of the main channel 16, downstream of the isolation valve50, before the channel splits into amplification channels 54 a and 54 b.

A particular arrangement of a preferred chamber in the cartridge, suchas the first and second amplification chambers or the first to fourthdetection chambers, may itself form an isolated inventive aspect of thecartridge, as described further in section 3, below.

It will be appreciated that the number of amplification chambers, andthe number of detection chambers in the exemplary cartridge may varydepending on the preferred implementation. Moreover, otherconfigurations of channels, chambers, valves and so on are possiblewithout departing from the scope of the invention, as defined by theclaims.

The physical structure and operation of the various components of theexemplary cartridge introduced above will now be explained withreference to FIGS. 2 to 10.

1.3 Physical Structure of an Exemplary Cartridge 1.3.1 Overview andExternal Features of the Exemplary Cartridge

An exemplary cartridge is shown in FIG. 2. As described above, thereader interacts with the cartridge through a plurality of interfaces.The interfaces shown in the exemplary cartridge 100 are: a pneumaticinterface 101; an electrical interface 102; a bypass valve interface103; and an isolation valve interface 104. Each of these interfaces isdescribed in more detail below. It will be appreciated that more orfewer interfaces could be provided, depending on the preferredimplementation.

Also provided in the cartridge, but not shown, is a thermal interface.The thermal interface allows the temperature of the amplificationchambers to be regulated to allow nucleic acid amplification to takeplace.

The exemplary cartridge 100 shown in FIG. 2 comprises an insertion end105 for insertion into the reader, and a non-insertion end 106.Proximate the non-insertion end 106 is a sample inlet 107 forintroducing a sample into the sample mixing chamber 10. In the exemplarycartridge, the sample will usually include cells, and the target nucleicacid (if present) can be extracted from these cells, but other fluidsamples such as swab eluate, urine, semen, blood, saliva, stool sweatand tears could be used in other implementations. The sample may beintroduced into the sample mixing chamber 10 through the sample inlet107 using a pipette, for example.

The exemplary cartridge 100 and reader are configured such that when thecartridge is inserted into the reader, all of the aforementionedinterfaces are actuatable by the reader. On the other hand, the sampleinlet 107 remains external to the reader such that a sample may beintroduced into the sample mixing chamber 10 whilst the cartridge isinserted into the reader.

The exemplary cartridge 100 shown in FIG. 2 further comprises a sampleindicator window 109, through which the sample indicator 12 is visibleto determine whether a sample has been introduced into the sample mixingchamber 10.

All of the pneumatic, mechanical and electrical interfaces in theexemplary cartridge 100 are located on the same face of the cartridge,in this case the top face 110. The thermal interface (not shown) isprovided on the bottom face of the cartridge. This simplifies the designof the reader, which may this provide the associated pneumatic,mechanical and electrical parts which interact with those interfaces inthe same region of the reader, thereby making best use of space. It alsoenables the thermal part of the reader to be provided away from thepneumatic, mechanical and electrical parts.

1.3.2 Internal Components of Cartridge

The exemplary cartridge 100 shown in FIG. 2 is formed from variouscomponents which shall now be described. FIG. 3 shows an exploded viewof the exemplary cartridge 100 of FIG. 2. The cartridge 100 comprises,from top to bottom, a housing 111, a blister sub-assembly 112, apneumatic foil 113, a pneumatic layer 114, a fluid layer 115 and afluidic foil 116. Also shown in FIG. 3 is an electrode layer 117, twofilters 118 and a plurality of absorbent pads 119, which will bedescribed in more detail below.

The housing 111 is manufactured from acrylonitrile butadiene styrene.The pneumatic and fluidic foils 113, 116 are manufactured from apolyethylene terephthalate/polypropylene composite. The pneumatic andfluidic layers 114, 115 are manufacture from polypropylene.

With the exception of the housing 111, filters 118 and pads 119, each ofthe components mentioned in the previous paragraph is adhered to itsadjacent component or components. Hence, the blister sub-assembly 112 isadhered to the pneumatic foil 113, which is adhered to the pneumaticlayer 114, which is adhered to the fluidic layer 115, which is adheredto the fluidic foil 116. The electrode layer 117 is adhered to fluidiclayer 115 also.

The adhesion of the layers to each other provides a series offluid-tight channels in the cartridge, together with associatedchambers, valves, pumps, bellows and other components. The channelspassing a liquid sample therethrough are liquid-tight and the channelspassing a gas therethrough are gas-tight. Optionally, all components areboth liquid tight and gas-tight. For example, recesses and openingsformed in one or both sides of the pneumatic and fluidic layers create,when sandwiched together and adhered to the pneumatic and fluidic foils,respectively, the shapes necessary to provide the aforesaid channels,chambers, valves, pumps, bellows and other components.

Each of the components referred to above in FIG. 3 will now be describedin more detail.

1.3.3 Housing 111

FIG. 4 shows housing 111 in more detail. As shown, housing 111 comprisesa generally rectangular upper surface 120 and walls 121 dependingtherefrom on all four sides (two of which are visible in FIG. 4). Aprincipal purpose of the housing 111 is to protect certain components ofthe cartridge, most notably the blister sub-assembly 112 and theisolation valve interface 104. It will therefore be noted that thehousing 111 is shorter than the pneumatic and fluidic layers 114, 115such that it overlies only a portion of those layers when the cartridge100 is assembled. In the exemplary cartridge 100, the pneumaticinterface 101, electronic interface 102, and bypass valve interface 103are not covered by the housing 111 to provide ease of access by thereader.

The upper surface 120 of the housing 111 has three apertures 122 a-ctherein, each having walls depending from the peripheries of theapertures to form, when the cartridge is assembled, three recesses. Thepurpose of the recesses is to house the blisters of the blistersub-assembly 112 such that the blisters may be accessed and pressed bythe reader, but are otherwise protected from accidental impact.Naturally, since the exemplary cartridge comprises three blisters, thehousing 111 comprises three corresponding apertures 122 a-c formingthree corresponding recesses. It will be appreciated that more or fewerblisters, apertures and recesses may be provided, depending on thepreferred implementation. Alternatively, the housing 111 could comprisea single aperture forming a single recess housing all availableblisters.

The side walls 121 of the housing 111 which run along the length of thehousing 111 between the insertion end 105 and the non-insertion end 106of the cartridge 100 comprise flanges 123 along at least a portion oftheir lower edges. The purpose of the flanges 123 is two-fold. Firstly,they comprise one or more windows 124 a-b for receiving a correspondingnumber of tabs formed in the pneumatic layer 114 to hold the cartridge100 together. Secondly, the flanges 123 are dimensioned so as toprotrude beyond the lower surface of the fluidic foil 116 when thecartridge is assembled, such that the fluidic foil 116 is suspendedabove a flat surface on which the cartridge 100 is placed. This preventsaccidental damage to the fluidic foil 1 16 which could otherwise result.

Although in the exemplary cartridge depicted in FIG. 4 flanges 123 areprovided along substantially the length of two opposing sides of thecartridge, it will be appreciated that flanges may be provided alongthree or four edges of the cartridge and still suspend the foil above aflat surface on which the cartridge is placed. Similarly, although thecartridge depicted in FIG. 4 shows flanges 123 extending alongsubstantially the entire length of the edge, a flange which extends onlypartially along an edge may be provided, or multiple flanges may beprovided along each edge.

The housing 111 further comprises, at the non-insertion end 106, a grip125 to facilitate insertion of the cartridge into and removal of thecartridge 100 from the reader by hand. The grip 125 comprises a seriesof ridges and grooves formed in the housing 111, but alternativestructures to increase friction, such as knurls, are also possible.

The housing 111 further comprises a sample inlet aperture 126 throughwhich a sample may be introduced into the sample mixing chamber 10 ofthe cartridge 100 using a pipette, for example. Surrounding the inletaperture 126 for a given diameter is a basin 127 recessed into the uppersurface 120 of the housing 111 to accommodate a certain amount ofspillage of the liquid sample. Whilst the basin 127 of the exemplaryembodiment is substantially flat, it may be sloped toward the inletaperture 126, such that any spillage drains through the inlet aperture126.

The exemplary housing 111 further comprises a plurality of cut-outs: afirst cut-out 128 forming the sample window 109, and a second cut-out129 to provide access to the isolation valve interface 104. As with therecesses which protect the blisters, by providing access to theisolation valve interface 104 only through a cut-out 129 in the housing111, the isolation valve interface 104 is protected to some extent fromaccidental impact, which could actuate the isolation valve and renderthe cartridge inoperable.

1.3.4 Blister Sub-Assembly 112

FIG. 5 shows the blister sub-assembly 112 in more detail. The blistersub-assembly 112 may be manufactured separately, during which theblisters are pre-filled with the liquid reagents necessary forconducting the preferred test, and subsequently adhered to the pneumaticfoil 113.

Blister sub-assemblies (or ‘blister packs’) are familiar to a skilledperson. A blister is a collapsible chamber for containing a liquid,which may be expelled from the blister by pressing on the blister andthereby collapsing it. In typical blister packs, the chamber of ablister is sealed by a foil or other frangible layer which ruptures oncethe pressure inside the chamber reaches a particular magnitude as theblister is collapsed.

In the exemplary cartridge, the blister sub-assembly 112 comprises threeblisters 130 a-c. These contain, respectively, the lysis buffer whichcomprises reagents capable of performing cell lysis, the wash buffer andthe elution buffer.

The exemplary blister sub-assembly 112 comprises a substrate 131 ontowhich the aforementioned blisters 130 a-c are formed by a deformablepolymeric layer which is shaped to provide the chambers. Three apertures132 a-c, corresponding to the three blisters 130 ac, pass through thesubstrate 132. Each of the apertures is covered by the deformablepolymeric layer forming the chamber, which thereby connects the apertureto the chamber but for a seal 133 a-c between the respective apertures132 a-c and chambers. Upon application of a suitable pressure on theblister 130 a-c, the seal 133 a-c breaks, thereby causing the liquidcontents of the blister to be ejected from the blister and to flowthrough the aperture 132 a-c in the substrate 131 out of the blistersub-assembly.

As shown, the seals 133 a-c at least partially surround the periphery ofthe chambers, where they meet the substrate 131. At the point in eachseal 133 a-c which is designed to break (thereby forming the liquidpassageway between the aperture 132 a-c and chamber), the seal 133 a-cmay be weaker than the rest of the periphery. This ensures that thecorrect part of the seal 133 a-c breaks when the suitable pressure isapplied.

The blisters may be collapsed by the reader when the cartridge isinserted therein. One or more mechanical actuators (such as a foot) maybe applied by the reader into the recess so as to collapse the blister.

The blister sub-assembly 1 12 further comprises two reference holes 134a-b configured to permit an assembly fixture to provide a reference tofacilitate positioning of the assembly during manufacture.

1.3.5 Pneumatic Layer 114

FIGS. 6A and 6B show the pneumatic layer 114 in more detail. FIG. 6A isa top view of the pneumatic layer and FIG. 6B is a bottom view. Thepneumatic layer 114 is comprised of a rigid plastic layer 135 which, incertain places, is overmoulded with a plurality of flexible membranes toform certain components when the cartridge is assembled. The flexiblemembranes are manufactured from a thermoplastic elastomer.

The rigid plastic layer 135 has a plurality of differently-shapedrecesses therein and apertures therethrough. In combination with thefluidic layer 115, certain recesses within, and/or apertures through,the rigid plastic layer 135 form a number of components, including: thesample mixing chamber 136; the waste chamber 137; the capture column138; the elution chamber 139; the first and second amplificationchambers 140 a-b; and the first to fourth detection chambers 141 a-d. Anaperture 142 is also provided to give access to the electrode layer 117.

In combination with the overmoulded flexible membranes and the pneumaticfoil 113, certain other apertures through the rigid plastic layer form anumber of other components, including: the upstream bellows valve 142;the bellows 143; a pneumatic interface 144; the downstream bellows valve145; the wash buffer inlet valve 146; the wash buffer air inlet valve146 a; the elution buffer inlet valve 147; the elution buffer air inletvalve 147 a; the waste chamber valve 148; the elution chamber valve 149;the isolation valve 150; the first and second amplification chamberinlet valves 151 a-b; and first and second amplification chamber outletvalves 152 a-b. A further aperture, in combination with an overmouldedflexible membrane (but not the pneumatic foil) forms a bypass valve 153.

With the exception of the isolation valve 150 and the bypass valve 153,the valves formed in the pneumatic layer are pneumatically-operablevalves. That is, each valve is operable to open and close a fluidicchannel in which the valve is located, and this valve is actuated byapplying a particular pressure to a pneumatic control line coupled tothe valve. The pneumatic control lines are coupled to the pneumaticinterface 144, to which the reader has access when the cartridge 100 isinserted therein. Hence, to actuate a given pneumatic valve, the readermerely applies an appropriate pressure to the pneumatic control lineassociated with that valve to open or close the valve.

The isolation valve 150 and the bypass valve 153 are also actuated bythe reader, but mechanically. Again, each valve is operable to open andclose a fluidic channel in which the valve is located, but the valve isactuated by applying one or more mechanical actuators (such as a foot)to the valve.

The pneumatic layer further comprises two reference holes 154 a-bconfigured to permit an assembly fixture to provide a reference tofacilitate positioning of the layer during manufacture. When thecartridge is assembled, the reference holes 154 a-b in the pneumaticlayer align with the reference holes 134 a-b in the blistersub-assembly.

The pneumatic layer further comprises apertures 155 a-c which, when thecartridge is assembled, line up with apertures 132 a-c passing throughthe substrate 131 of the blister sub-assembly (through the pneumaticfoil, as described below).

1.3.6 Pneumatic Foil 113

FIG. 7 shows the pneumatic foil 113 in more detail. As explained above,the pneumatic foil 113 is adhered to the upper surface of the pneumaticlayer 114, thereby fluidly sealing channels, chambers, valves, pumps,bellows and other components formed therein. Thus, for the most part,the pneumatic foil 113 is a generally rectangular and planar foil sheetso as to provide an effective seal. Beneficially, the pneumatic foil 113is inert such that is does not react with the reagents which movethrough the pneumatic layer 114.

However, the pneumatic foil 113 does not overlie the entire pneumaticlayer 114. In particular, the pneumatic foil 113 does not overlie thesample mixing chamber 136 or the waste chamber 137 at the non-insertionend 106 of the cartridge 100, or the bypass valve 153 at the insertionend 105. Moreover, the pneumatic foil 113 comprises cut-outs 156, 157,such that it does not overlie the isolation valve 150 or the pneumaticinterface 144, respectively.

The pneumatic foil 113 further comprises three apertures 158 a-c which,when the cartridge 100 is assembled, line up with apertures 132 a-cpassing through the substrate 131 of the blister sub-assembly and 155a-c passing through the pneumatic layer 114. The apertures 158 a-cpermit the liquid reagents within the blisters to pass to the pneumaticlayer 114, and thence to the fluidic layer 115 through apertures 155a-c.

The pneumatic foil 113 comprises two reference holes 159 a-b configuredto permit an assembly fixture to provide a reference to facilitatepositioning of the layer during manufacture. When the cartridge isassembled, the reference holes 159 a-b in the pneumatic foil align withthe reference holes in the other layers.

The pneumatic foil is a composite foil manufactured from a layer ofpolyethylene terephthalate, to provide strength, with a layer ofpolypropylene on top to provide an inert material for contacting theliquid sample and buffers, and also to enable the foil to be heat sealedto the pneumatic layer (also manufactured from polypropylene.

1.3.7 Fluidic Layer 115

FIGS. 8A and 8B show the fluidic layer 115 in more detail. FIG. 8A is atop view of the pneumatic layer and FIG. 8B is a bottom view. Thefluidic layer 115 is comprised of a rigid plastic layer 160. Asexplained previously, the top side of the fluidic layer 115 (not shown)is adhered to the bottom side of the pneumatic layer 113 (see FIG. 5B)such that the various channels, chambers, valves, pumps, bellows andother components formed by a combination of the pneumatic and fluidiclayers are aligned.

As with the rigid plastic layer 135 of the pneumatic layer 113, therigid plastic layer 160 of the fluidic layer 1 15 has a plurality ofdifferently-shaped recesses therein and apertures therethrough. Incombination with the pneumatic layer 113 and the fluidic foil 116,certain recesses within, and/or apertures through, the rigid plasticlayer 160 forms certain components, including: the sample inlet chamber136; the capture column 138; the elution chamber 139; the first andsecond amplification chambers 140 a-b; and the first to fourth detectionchambers 141 a-d. the upstream bellows valve 142; the bellows 143; thepneumatic interface 144; the downstream bellows valve 145; the washbuffer inlet valve 146; the wash buffer air inlet valve 146 a; theelution buffer inlet valve 147; the elution buffer air inlet valve 147a; the waste chamber valve 148; the elution chamber valve 149; theisolation valve 150; the first and second amplification chamber inletvalves 151 a-b; and first and second amplification chamber outlet valves152 a-b. An aperture 161 is also provided to give access to theelectrode layer 117.

Moreover, in combination with the fluidic foil 116 (but not thepneumatic layer 114), recesses in the fluidic layer 115 also providesthe coarse filter 162, the convoluted mixing channel 163, and aplurality of channels which, when the cartridge is assembled, connectthe aforementioned components together to enable passage of the liquidsample and liquid reagents through the cartridge, and facilitatepneumatic actuation of the valves, pumps, bellows and other components.

The fluidic layer comprises two reference holes 164 a-b configured topermit an assembly fixture to provide a reference to facilitatepositioning of the layer during manufacture. When the cartridge isassembled, the reference holes 164 a-b in the fluidic layer align withthe reference holes in the other layers.

As mentioned above, channels are formed between the pneumatic interfaceand the various valve and bellows described above. In the exemplarycartridge, the pneumatic interface comprises 11 ports which areconnected to the various components as follows.

-   -   Port 1 bellows    -   Port 2 upstream bellows valve first and second amplification        chamber inlet valves first and second amplification chamber        outlet valves    -   Port 3 downstream bellows valve    -   Port 4 wash buffer inlet valve    -   Port 5 wash buffer air inlet    -   Port 6 wash buffer air inlet valve    -   elution buffer air inlet valve    -   Port 7 elution butter air inlet    -   Port 8 elution buffer inlet valve    -   Port 9 reference pressure line    -   Port 10 elution chamber valve    -   Port 11 waste chamber valve

It will be understood that whilst various inventive aspects of theexemplary cartridge may be implemented using specific ones of theconnections listed above (in particular, the first and secondamplification chamber inlet and outlet valves being connected to asingle port; and the wash and elution buffer air inlets being connectedto a single port); the precise configuration listed above is notessential.

1.3.8 Fluidic Foil

FIG. 9 shows the fluidic foil 116 in more detail. As explained above,the fluidic foil 116 is adhered to the lower surface of the fluidiclayer 115, thereby fluidly sealing channels, chambers, valves, pumps,bellows and other components formed therein. Thus, for the most part,the fluidic foil 116 is a generally rectangular and planar foil sheet soas to provide an effective seal. Beneficially, the foil 116 is inertsuch that is does not react with the reagents which move in thepneumatic layer.

However, the fluidic foil 116 does not overlie the entire fluidic layer115. In particular, the fluidic foil 116 does not overlie the detectionchambers 141 a-d at the insertion end 105.

The fluidic foil 116 comprises two reference holes 165 a-b configured topermit an assembly fixture to provide a reference to facilitatepositioning of the layer during manufacture. When the cartridge isassembled, the reference holes 165 a-b in the fluidic foil aligns withthe reference holes in the other layers.

The fluidic foil is a composite foil manufactured from a layer ofpolyethylene terephthalate, to provide strength, with a layer ofpolypropylene on top to provide an inert material for contacting theliquid sample and buffers, and also to enable the foil to be heat sealedto the fluidic layer (also manufactured from polypropylene.

1.3.9 Electrode Layer 117

Finally, FIG. 10 shows the electrode layer 117 in more detail. Asexplained above, the electrode layer 117 is adhered to the fluidic layer115. The electrode layer 117 comprises four sets of detection electrodes166 a-d. Each set of detection electrodes 166 a-d comprises first tothird electrical contacts 168 a-d which couple with correspondingelectrical contacts in the reader when the cartridge is insertedtherein. Preferably, the electrical contacts are made of silver tooptimise the electrical connection. Preferably electrodes which aresilver plated with silver chloride are used to ensure a the optimalgalvanic behaviour.

Each set of detection electrodes 166 a-d comprises a working electrode169 a-d; a counter electrode 170 a-d and a reference electrode 171 a-d.Each of the electrodes is coupled to a respective electrical contact.Each set of detection electrodes 166 a-d also comprises a dielectric 172a-d covering the interface between the electrodes and the respectiveelectrical contacts.

Electrochemical signalling may be used to indicate the presence ofgenetic or immunohistochemistry targets in a sample. The sample isprocessed to form an electrolyte which, in practice, may be held in acell comprising a set of detection electrodes. Upon application of apotential difference across the work electrodes 169 a-d and counterelectrodes 170 a-d in the cell, some compounds in an electrolyte willhave a natural tendency to migrate to the electrodes and swap electrons,resulting in a tiny current. All combinations of soluble compounds havesome electrochemical activity, and the rate at which this activityoccurs enables measurement of the quantity of those compounds. Thus, thepresence of different compounds in the sample may be measured bysearching for characteristic features of their redox electrochemistry.In particular, the sample may be processed to include labels that areselected compounds that are present if and only if the sample containstarget molecules.

In the exemplary cartridge, the electrodes 166 a-d are arranged suchthat a liquid sample within the first to fourth detection chambers 141a-d comes into contact with the first to fourth sets of electrodes 166a-d.

A voltage sweep is applied between the working electrodes 169 a-d andcounter electrodes 170 a-d by the reader.

The current flowing at any given thus-corrected potential difference ismeasured and provides the signal that is indicative of the labelcompounds in the sample. Conventionally, the counter electrode isexcessively large so that the reaction at this electrode does not limitthe current flowing as a result of the reaction of the workingelectrode, which is the one of interest.

1.3.10 Bypass Valve (Mechanical Valve)

FIG. 43 shows a first embodiment of a valve D100 of the exemplaryfluidic cartridge. The valve is mentioned above as being a bypass valvefor reasons that will become apparent. It is also referred to herein asthe mechanical valve.

The valve D100 comprises a valve cavity D101 and a flexible valvemembrane D105 provided within valve cavity D101. The valve cavity D101may be formed in a single polymer layer or may be comprised of aplurality of layers, such as the housing 111, pneumatic layer 114, andfluidic layer 115 of the exemplary cartridge 100. The valve membraneD105 is formed within the valve cavity D101 and may be over-moulded ontothe pneumatic layer, as explained above.

The valve chamber D101 has side walls D130, a floor D132 and is open atthe top. The valve chamber D101 comprises first and second openingsD102, D103 in the floor D132 connected to first and second passageways,D112, D113 formed in the fluidic layer 115. First and second openingsD102, D103 may be located on first and second raised portions of valvechamber D101 to form first and second valve seats.

Between the flexible membrane D105 and the floor D132 of the valvecavity D101, a valve chamber D115 is defined. The valve chamber istherefore fluidly connected to the first and second openings D102, D103.

Valve membrane D105 comprises a first valve membrane portion D122 and asecond valve membrane portion D123.

The first valve membrane portion D122 is movable between an openposition, in which it is spaced apart from the first opening D102 andpermits fluid to flow between the first passageway D112 and the valvechamber D115, and a closed position, in which it seals against the firstopening D102 and prevents any fluid flow between the first passagewayD112 and the valve chamber D115.

Similarly, the second valve membrane portion D123 is movable between anopen position in which it is spaced apart from the second opening D103and permits fluid to flow between the second passageway D113 and thevalve chamber D115, and a closed position, in which it seals against thesecond opening D102 and prevents any fluid flow between the secondpassageway D113 and the valve chamber D115.

It will therefore be appreciated that the valve chamber D115 has a firstvolume, V1, when the first and second valve portions are in their openpositions; a second volume, V2, when one of the first and second valveportions is in its open position and the other is in its closedposition; and a third volume, V3, when the first and second valvepositions are in their closed positions. It will be appreciated thatV1>V2>V3. Volume V3 is ideally as close to zero as possible.

The first and second valve membrane portions D122, D123 are actuatableindependently of one another. For instance, when the valve is used inthe exemplary cartridge and when the cartridge is inserted into areader, the reader may apply first and second mechanical actuators, suchas feet, to actuate the first and second valve membrane portions D122and D123 independently. This is advantageous in a sealed system, such asthe back end of the exemplary cartridge where there is a criticalpressure to be maintained on one side of the valve. In this case thevalve seat corresponding to the first and second channels can beactuated first while keeping the valve cavity open to the bypasschannel, to avoid pressurising the first and second channels ordisplacing the liquid therein.

Referring now to FIG. 44, a second embodiment of a valve D200 isillustrated. The second embodiment is identical to the first (and likereference numerals refer to similar features), except that a thirdopening D204 is provided in the floor of the valve cavity in addition tothe first and second openings D202, D203. The third opening D204 isconnected to a third passageway D214 and is adapted to be sealed by thesecond valve membrane portion D223, to prevent fluid moving between thethird passageway D214 and the valve chamber D250.

In the embodiment shown in FIG. 44, second and third openings D203, D204are located on second and third raised portions D243, D244. However, itwill be appreciated that second and third opening may be located on asingle raised portion D343, or that they may be located on a regionsubstantially flush with the rest of the valve cavity floor.

As shown, the second and third openings D203, D204 are spaced apart bydistance b. First opening D202 is spaced apart from the second openingD203 by distance a. The distance a between first and second openingsD202, D203 is greater than the distance b between second and thirdopenings D203, D204. This is convenient to enable the second and thirdopenings D203, D204 to be sealed by the second membrane portion D223 andthe first opening D202 to be sealed by the first membrane portion D222.

Although in the first and second embodiments illustrated in FIGS. 43 and44, the valve is shown as having two or three openings, it will beappreciated that four or more openings may be provided. The openings maybe grouped in any manner so as to be sealed by the first membraneportion or the second membrane portion, depending on the preferredimplementation. It will also be appreciated that, although in theembodiments shown in the drawings, the valve membrane is shown to havefirst and second valve membrane portions, it possible that the valvemembrane may have three or more portions, each adapted to seal one ormore openings and each adapted to be independently actuatable.

As described above, the first valve membrane portion D222 and the secondvalve membrane portion D223 may be mechanically actuated by first andsecond mechanical actuators D232, D233 which could, for instance, beprovided in a reader (not shown). The first mechanical actuator D232 isconfigured to be movable from a first position in which it is spacedapart from the first valve membrane portion D222 and a second positionin which it presses first valve membrane portion against opening D202,thereby sealing the opening. Similarly the second mechanical actuatorD232 is configured to be movable from a first position in which it isspaced apart from the second valve membrane portion D223, to a secondposition in which it presses second valve membrane portion D233 againstthe second opening and third openings D203, D204.

The valve may be configured such that the mechanical actuators D232 andD233 may contact substantially all of the valve membrane D205.Alternatively, the valve may be configured such that the mechanicalactuators D232 and D233 may only contact a portion of valve membraneD105.

Referring now to FIGS. 44 and 45, it can be seen that by positioning thesecond and third openings relatively close together, the second valvemembrane portion D223 may be actuated to effectively seal the second andthird openings D203, D204 without requiring a large surface area tocontact the valve membrane D205. In contrast, the relatively largedistance between the second and first openings D202, D203 allows thesecond portion of valve membrane D223 to be depressed by the secondbiasing means without significantly depressing the first valve membraneportion D222.

Preferably, valve membrane D205 is formed of resiliently deformablepolymer such that the valve is biased into the first position.Preferably, the valve membrane D205 has a thickness of at least 0.25 mm,most preferably a thickness of around 1 mm. This ensures that the valvemembrane is thick enough to provide compliance for an effective sealover the openings. By moving the biasing means D232, D233 from thesecond position to the first position, biasing means D232, D233 nolonger press valve membrane D105 against openings D202, D203, D204 andthe valve returns to the open position.

An implementation of the valve D200 will be explained with reference toFIG. 46. In particular valve D200 is used as a bypass valve 68 in theback end of the exemplary fluidic cartridge 100 discussed above.

FIG. 47 shows following features of the exemplary cartridge 100: theelution branch 16 b of the main channel 16; the isolation valve 50; themixing channel 52; the first and second PCR channels 54 a, 54 b and thefirst and second bypass channels 66 a, 66 b. Certain features present inthe exemplary cartridge 100 are omitted from FIG. 47 for clarity.

As illustrated in FIG. 46, the first and second bypass channels 66 a, 66b are respectively connected to the second and third openings D203, D204of the bypass valve D200. The first opening 202 and first passagewayD212 are coupled to the elution branch 16 b of the main channel 16downstream of the isolation valve.

As described above, the back end of the exemplary fluid cartridge formsa closed system when the isolation valve 50 is closed. Hence, a firstadvantage of using the bypass valve D200 in the valve system D500 shownin FIG. 47 is that it can be used to depressurise the back end after thetest is complete. This may occur as follows. Once the liquid sample hasbeen pumped into the detection chambers (not shown) of the exemplarycartridge 100, the isolation valve may be closed to form a closed systemin the back end. However, at a suitable point before the isolation valve50 is closed, the first, and preferably second flexible membraneportions D222, D223 may be pushed by mechanical actuators into theirclosed positions, thereby decreasing the volume within the valve chamberD250.

When the volume of the valve chamber D250 is below its maximum (forinstance when one or both of the flexible membrane portions D222, D223is in its closed position), the isolation valve may be closed, therebyforming a sealed system in the back end. Once the isolation valve isclosed, the flexible membrane portions D222, D223 may be returned totheir open positions, thereby increasing the volume of the valve chamberD250 to its maximum.

In one example, when valve D200 is in the open position and valvemembrane D205 is spaced apart from openings D203, D204, as shown in FIG.44, the volume of valve chamber D250 is V. When valve D200 is in theclosed position, as shown in FIG. 46, the volume of valve cavity D250 issubstantially zero.

Hence, when valve D200 is the open position, the volume of the valvesystem D500 is:

Vopen=Vcavity+Vnetwork.

When valve D200 is in the closed position, the volume of the valvesystem D500 is:

Vclosed=Vnetwork.

Providing the isolation valve is open when valve D200 is closed, anamount of fluid equal to Vcavity will be displaced outside the networkof channels (upstream of the isolation valve) and there will be anamount of fluid equal to Vnetwork left in the network of channels(downstream of the isolation valve).

When isolation valve 50 is closed, the system becomes a closed systemand the quantity of fluid in that system is fixed. When valve D200 isthen reopened after the isolation valve has been closed, the volume ofthe system returns to Vopen. Since Vopen>Vclosed, a negative pressure iscreated in the system. This negative pressure reduces the risk ofleakage of the cartridge. It will be appreciated that if this reductionin pressure is large enough, it is possible to create a negativepressure in a system, even where the system is initially slightlypressurised.

By closing valve D200 to reduce the volume of the system, closing theisolation valve to close the system, and then opening valve D200 toincrease the volume of the system, it is possible to achieve a negativepressure within the back end of the exemplary cartridge. Preferably, thechange in the volume of valve cavity D250 is large enough to effect asignificant pressure change in the fluidic network. Although in theembodiments shown in the drawings, valve chamber is shown to have two orthree openings, it will be appreciated that this method ofdepressurising a system will work with any number of openings.

As described above, the first and second bypass channels 66 a, 66 b maybe used to remove excess fluid sample from the first and second fluidpathways through the first and second PCR channels 54 a, 54 b. Thus, thefirst bypass channel 66 a is coupled to the first fluid pathway in thefirst PCR channel 54 a and the second bypass channel 66 b is coupled tothe second fluid pathway in the second PCR channel 54 b.

At an appropriate point in the test, it is necessary to close the bypassvalve D200. However, when closing the bypass valve, there is a risk thatthe pressure change caused by the membrane sealing against the secondand third openings D203, D204 will push fluid in the bypass channels 66a, 66 b back toward the PCR channels 66 a, 66 b, particularly if fluidis unable to escape elsewhere in the system. This is undesirable. Hence,a second advantage of using the bypass valve D200 in the valve systemD500 shown in FIG. 47 is that the pressure change causing such backflowcan be mitigated.

By using a valve D200, a first step of applying a force to the secondvalve membrane portion may be carried out to seal the second valvemembrane portion against the second and third openings of the valvechamber. FIG. 45 shows the valve D200 in an intermediate positionwherein second and third openings are sealed by second valve membraneportion D223 whilst the first opening D202 remains open. A second stepis then performed which comprises applying a force to the first valvemembrane portion D222 to seal the first valve membrane D222 portionagainst the first opening D202 in the valve chamber D101.

By closing the second and third openings before closing the firstopening in the valve chamber, it is possible to avoid pressurising thesecond and third passageways excessively, and in fact to minimise theback flow into the first and second bypass channels.

Although the method described above refers to a valve having first,second and third openings, it will be appreciated that this method maybe adapted for valves having two or four or more valves arranged in twogroups, wherein the first group of valves is sealed by the first valvemembrane portion, and the second group of valves may be sealed by thesecond valve membrane portion. In this context, it is intended that agroup refer to one or more valves.

Preferably, in embodiments having four or more openings, the first valvemembrane portion is configured to seal the first opening and the secondvalve membrane portion is adapted to seal any subsequent openings.Additionally, the opening of the valve chamber may be located on araised portion of the valve chamber to create a raised valve seat. Eachraised portion may comprise multiple openings, or each opening may beprovided with its own raised portion. Alternatively some or all of theopenings may not be located on a raised portion.

The method operation of the exemplary cartridge introduced above willnow be briefly explained.

1.4 Method of Operation of the Exemplary Cartridge

1.4.1 The front end

As described above, a fluid sample (such as a urine sample) isintroduced into the sample mixing chamber 10 using a pipette. A portionof the sample passes to the sample indicator 12 to show that a sample ispresent in the sample mixing chamber.

Once the cartridge 100 with a sample in the mixing chamber 10 isinserted into a reader, and the reader is activated, the test maycommence. Firstly, the reader will apply a mechanical actuator (such asa foot) to collapse the lysis buffer blister 14. In doing so, the lysisbuffer will be expelled into the sample mixing chamber 10 where it willmix with the sample.

The bellows 20 and its valves 22 a-b then moves the liquid sample andlysis buffer back and forth into the sample mixing chamber 10 so as tomix the lysis and sample and to rehydrate the internal control.Following the mixing step, incubation of the sample and lysis bufferoccurs to allow cell lysis to take place.

The bellows 20 and its valves 22 a-b will then commence operation topump the sample from the sample mixing chamber 10, into the main channel16, through the coarse filter 18 and toward the capture column 24.Within the capture column 24 nucleic acids are specifically bound to afilter in the capture column on the basis of their size and charge. Theunwanted liquid sample passes through to the waste chamber 38.

Once the unwanted liquid sample has passed to the waste chamber 38,leaving the nucleic acids bound to the capture column 24, the readerapplies a mechanical actuator (such as a foot) to collapse the washbuffer blister 30. In doing so, the wash buffer will be expelled intothe first branch channel 26, and thence into the main channel 16. Thewash buffer will be flushed into the waste chambers using air from thewash and/or elution buffer air inlets.

Once the wash sample has passed to the waste chamber 38, leaving onlythe bound and purified nucleic acids in the capture column 24, thereader applies a mechanical actuator (such as a foot) to collapse theelution buffer blister 32. In doing so, the elution buffer will beexpelled into the second branch channel 28, and thence into the mainchannel 16, through the capture column 24 to elute the nucleic acidsfrom the capture column, to the elution chamber 46.

The sample settles in the elution chamber 46 allowing bubbles todisperse before entering the amplification chambers.

1.4.2 the Back End

A controlled pressure is applied to transfer fluid from the elutionchamber through the isolation valve 59 to the amplification chambers 56a-b. Any excess liquid sample may be removed from the fluid pathwaythrough the bypass channels 68. In the nucleic acid amplificationchambers 56 a-d the nucleic acid or acids of interest, if present, isamplified such that it is present at a detectable level. The controlnucleic acid is also amplified such that it is present at a detectablelevel. As mentioned above, any nucleic acid amplification method may beused. Where PCR is used, primers specifically hybridise to the nucleicacid of interest and are extended by a thermostable polymerase such asTaq polymerase via the addition of dNTPs to the 3′ end of each of theprimers. Before the PCR starts, the valves at the entry and exit to thePCR chambers are closed, isolating the sample, and the detectionchambers are vented via the mechanical valve back to the elution chamberwhich is now vented to atmosphere.

A controlled pressure is applied to transfer the fluid from theamplification chambers to the detection chambers. In the detectionchambers, the target probe specifically hybridises to the targetamplified nucleic acid of interest and the control probe specificallyhybridises to the amplified control nucleic acid. The nucleasehydrolyses the target and control probes following hybridisation of theprobes to the amplified nucleic acid. The hydrolysis of the target andcontrol probes frees the labels from the probes causing a detectablechange in the signal from the labels to occur.

Once the liquid sample occupies the detection chambers, the readerapplies a mechanical actuator to the isolation valve 50 to close thevalve and isolate the liquid sample in the back end of the device.

The electrodes provide a potential difference across the at least onedetection chamber. Depending on the state of the label (i.e. whether itis attached to the full length probe or the probe has been hydrolysedand it is free or attached to a single nucleotide or short part of theprobe), the diffusion rate to the electrode will differ consequently thecurrent that is able to flow through the detection chamber will differ.The electrodes therefore allow detection by the reader of the change inthe signal from the label which results from hydrolysis of hybridisedprobe.

2. The Exemplary Cartridge Reader 2.1 Overview

The exemplary cartridge reader comprises a housing containing: a drawerfor inserting a cartridge in to the reader; first and second clamps,adapted to receive and hold the exemplary cartridge therebetween; threeblister actuators for actuating three collapsible blisters comprised inthe exemplary cartridge; a pneumatics module, comprising a pneumaticsinterface for interfacing with the pneumatic ports on the exemplarycartridge and at least one pump for providing an appropriate pressure tothe pneumatics interface; a thermal module, comprising three thermalstacks for controlling the temperature of three zones within theexemplary cartridge; an electrical interface for electrically connectingto the electrodes comprised in the exemplary cartridge; an isolationvalve closing mechanism for actuating a latching isolation valve in theexemplary cartridge; and a mechanical valve actuator for actuating thebypass valve comprised in the exemplary cartridge.

2.2 General arrangement and thermal zones

FIGS. 11a and 11b show an external view of a cartridge reader 200 inwhich the present invention may be implemented. The cartridge reader 200comprises an outer housing 201, a cartridge drawer 202, a touchscreendisplay 203, and a power button 204.

The housing takes the form of a triangular prism having a front face205; a rear face 206 and a base (not shown) flanked by first and secondsides 206 a, 206 b. The front and rear faces of the housing meet at atop edge 207.

Vents are provided in the housing for dissipating heat, and include afirst vent 208 in the rear face 206 proximate the top edge 207; secondvents 209 a, 209 b in the base along bottom edges of the housing, morespecifically between the base and sides (i.e. where the first and secondsides 206 a, 206 b meet the base); and third vents 210 a, 210 b in thefirst and second sides 206 a, 206 b, more specifically between the firstand second sides 206 a, 206 b and each of the rear face 206 and the base(i.e. where the first and second sides 206 a, 206 b meet the base andthe rear face).

FIGS. 12a and 12b are exploded diagrams of the housing of FIGS. 11a and11b . FIG. 12a illustrates the parts of the housing which form the base211 and rear face 206, which is an integral piece formed (for example)by injection moulding. As shown, within the housing are supportstructures 212 a, 212 b that provide support for internal components ofthe housing and also contribute to the thermal management within thehousing, as described in more detail below.

FIG. 12b illustrates the parts of the housing which form the front face204 and the first and second sides. Each of these parts is formedseparately, and is mounted to the remaining components of the housingusing suitable fixing means such as screws.

FIG. 13 shows the bottom of the base 211 of the housing; second andthird vents 209 a, 209 b, 210 a, 210 b are clearly visible.

An access panel 214 is provided in the base of the reader 200. Theaccess panel is fixed to the base 211 using suitable fixing means suchas screws to allow easy access to the internal components of the reader200, as described in more detail below.

FIG. 14 shows some of the principal subsystems of the reader 200.Specifically the reader comprises a power control module 215 forreceiving power from an external power supply and distributing the poweramongst the components of the reader; an electronics control module 216for managing the electrochemical detection and analysis of a samplewithin a cartridge; a thermal module 217 for managing thermal energysupplied to a cartridge during a test; a pneumatics block 218 formanaging the delivery of fluid pressure to a cartridge during a test;and a cartridge handling module 219 for receiving the cartridge andclamping it within the housing. Other components illustrated include thetouchscreen and a cartridge 100. Other principal subsystems of thereader 200 that are not shown in the figure are the blister actuatorsystem and the mechanical valves actuator system.

FIG. 15 shows the internal components of the reader organised onto sixboards—a main board 220, a thermal board 221, a power board 222, apotentiostat pogo pin board 223, a connector board 224 and a sensorboard 225.

The main board 220 provides overall control of the reader. It comprisesa CPU module 225 for processing and generating instructions forcontrolling the remaining components of the reader. The main board 220also comprises four signalling LEDs 227 and a buzzer 228 for indicatingvisually and aurally the status of the device; I/O ports including anEthernet port 229 and three USB ports 230; sensors including threeoptical sensors 231 and four pressure sensors 232. The main board isalso coupled to the LCD touch screen 233 which provides user input forthe CPU.

The thermal board 221 comprises potentiostat circuits 234 for applyingvoltage and detecting current in a detection chamber in a cartridge. Thepotentiostat circuits 234 are coupled to the main board via an ADC 235a. The potentiostat circuits 234 are also coupled to the potentiostatpogo pin board 223, which comprises twelve pogo pins 236 for coupling toelectrical contacts on the cartridge.

The thermal board also comprises peltier drivers 237 which drive thepeltier devices on the connector board 224 for applying thermal energyto the cartridge. The peltier drivers 237 are coupled to the main boardand to the sensor board via an ADC 235 b.

The power board comprises connectors to 7 stepper motors, 18 valves, 2pumps, 1 solenoid and 1 electromagnet. The power board is also coupledto the barcode engine 238.

The connector board comprises connectors to three peltier devices andthree fans. The sensor board comprises six peltier temperature sensorsconnected to the thermal board via an ADC 235 c.

DC power in 239 is provided on the thermal board 221, and it distributedfrom the thermal board across 24 v power lines 240 a-c to the mainboard, power board, connector board and sensor board. Signal lines 241a, 241 b interconnect the main board and the thermal and power boards,whilst signal lines 241 c, 241 d interconnect the main board and thethermal and power boards.

FIG. 16 shows a cross section of the reader 200. As can be seen, thereader is partitioned into three thermal zones by thermally insulatingwalls 245. A first zone 242 is provided at the base of the reader towardthe front face 205. The first zone contains the thermal module 217,thereby preventing heat dissipated from the thermal module from enteringthe second or third zones. In particular, the first zone comprises thethree peltiers, three fans and six peltier temperature sensors connectedto the connector board.

A second zone 243 contains power control module 215, electronics controlmodule 216, pneumatics block 218 and cartridge handling module 219, aswell as the blister actuator system and the mechanical valves actuatorsystem.

A third zone 244 is provided toward the rear face and elevated above thebase but spaced apart from the top of the reader. The third zone, whichis the coolest zone, comprises the main board.

Each of the first second and third zones comprises a fan for drawing airinto the zone before it is expelled from a vent.

2.3 Cartridge Handling

FIGS. 17a and 17b show the drawer 202 of the cartridge reader in moredetail. As shown in FIGS. 17a and b , the drawer 202 comprises a frontportion 250, a rear portion 251, and first and second side portions 252a, 252 b. The first and second side portions connect the front and rearportions, thereby defining the periphery of a cartridge receiving tray253. A cut-out 254 is provided in tray 253, such that a cartridgeinserted into the tray is support around its periphery by the tray 253,whilst substantially all of a lower surface of the cartridge 100 isaccessible through the cut-out 254. A vertical rib 256 runs along one ofthe first and second side portions 252 a, 252 b. A drawer spring 258,which biases the drawer into an open position relative to the upperclamp is provided above one of the first and second side portions 252 a,252 b towards the rear of the drawer 202. As shown in FIG. 17a , thecartridge 100 and the cartridge receiving tray 253 comprise anasymmetrical design such that the cartridge 100 can only be placed inthe drawer 202 in the orientation shown in FIG. 17a . In the case of theexemplary cartridge described above, this means that the sample inletport is located at the front of the drawer 202, whilst the detection andsample processing chambers are located at the rear of the drawer 202.

Referring now to FIGS. 18a and 18b , the drawer 202 is slidably mountedin the upper clamp 260. The vertical rib 256 engages a groove 262provided in the upper clamp 260, thereby restraining movement of thedrawer 202 in a first direction relative to the upper clamp 260. Thegroove 262 narrow towards the rear (for example in the last 20 mm of thedrawer closure), thereby tightly aligning the drawer 202 within theupper clamp 260. Adjacent to the rib 256, the drawer 202 also includes aflag feature (not shown) that is viewed by two optical sensors (notshown) rigidly attached to the upper clamp 260. These provide signalsthat inform the reader's control system whether the drawer 202 is openor closed.

A drawer latching mechanism is provided between the drawer 202 and theupper clamp 260. Turning to FIG. 19, the drawer latching mechanismcomprises a latch pin 264, provided in the upper clamp 260, which alignswith a recess 266 provided in the drawer 260 when the drawer is closed.The latch pin 264 is biased by biasing means (not shown) into a positionin which it engages the latch recess 266. A solenoid (not shown) isprovided in the upper clamp 260, which can be selectively activated toengage the latch pin 264.

The latching mechanism shown in FIG. 19 holds the drawer 202 closedduring a test carried out by the reader 200 and, additionally, whilstthe reader is in its inactive state. At an appropriate point during thetest cycle (i.e. when an exemplary cartridge 100 is to be inserted orremoved), the reader 200 will momentarily active the solenoid, therebyretracting the latch pin 264 from the recess 266. Once latch pin 264 hasbeen withdrawn from the recess 266, drawer 260 opens automatically underthe influence of the drawer spring 258. A ramp 270 is provided at therear of the drawer 202 adjacent to the recess 266. As a user pushes thedrawer 202 closed, the latch pin 264 rides over the ramp 270 andreengages the recess 266, thereby resetting the drawer latch mechanismand latching the drawer in its closed position.

Turning now to FIG. 20, the drawer 202 and the upper clamp 250 arepositioned at an angle of 5 degrees relative to a horizontal defined bya surface on which the reader is placed. In practice, this means thatfor an exemplary cartridge 100 inserted into the reader, the sampleinlet is located above the detection and sample processing chambers.

FIGS. 21a and 21b show schematic diagrams of the upper clamp 260 and thelower clamp 288 respectively. As shown in these figures, the clampscontain a number of screw-in inserts that provide fixing points formating parts. To prevent the risk of the inserts pulling out, the designuses captive inserts that are screwed in from the opposite side to thefixing.

Referring now to FIG. 21a , the upper clamp 260 is fixedly mountedwithin the cartridge reader 200 comprises a cartridge receiving region271, a rear portion 272 a and two extending side portions 272 b-c. Thecartridge receiving region 272 comprises: a pneumatics interface 274; anelectrical interface 276; first, second and third blister actuators 278a-c; a mechanical valve actuator 280; and an isolation valve actuator282. First, second and third clamp actuators 284 a-c are fixed withinthe rear and side portions of the upper clamp 260. Each clamp actuatorcomprises a lead-screw 291 a-c driven by stepper motor (not shown).Adjacent each clamp actuator 284 a-c, the upper clamp 260 comprises atleast one hard stop 286.

Referring now to FIG. 21b , the lower clamp 288 is movable within thecartridge reader relative to the upper clamp and comprises a centralraised portion 289, a rear portion 290 a two side portions 290 b-cextending either side of the central raised portion 289. The raisedcentral portion 289 is dimensioned to fit through the cut out 254provided in the drawer 202. First second and third clamp actuator nuts292 a-c are mounted in the rear portion and the first and second sideportions of the lower clamp 288 respectively. Each of the three clampactuators nuts 292 a-c is threadedly engaged with the correspondingclamp actuator 284 a-c mounted in the upper clamp 260. In addition tothe above, the lower clamp 288 comprises first, second and third heatstacks 287 a-c, which will be described in more detail hereafter.

FIG. 22 show a cross-section of a cartridge 100 clamped between theupper and lower clamps.

The three clamp actuators 284 a-c connecting the upper clamp 260 to thelower clamp 288 are clearly shown.

Two static alignment shafts 294 a-b are fixedly connected to the lowerclamp 288 and pass through two corresponding apertures 296 on first andsecond side portions of the upper clamp 260. However, a skilled personwill understand that more than two alignment shafts may be provided. Agantry 298 is fixedly connected between the two alignment shafts 296above the upper clamp 260. First and second priming springs 300 a-b areprovided between the upper clamp 260 and the gantry 298, preferablyaround each of the static alignment shafts 294. Two location pins 302protrude from a lower surface of the upper clamp 260, for aligning withtwo corresponding recesses in an exemplary cartridge 100 inserted intothe reader.

Fixed bushes 304 in the lower clamp 288 ensure that the alignment shafts294 remain perpendicular to the lower clamp 288 whilst the clampactuator nuts 292 a-c are allowed to float in a captive space betweenthe lower clamp 288 and its support bracket 306. At least one mouldedstop feature (not shown) in the lower clamp 288 prevents rotation of theclamp actuator nuts 292 a-c and the design ensures that the nuts 292 a-conly experience vertical forces, not rotational forces that could shearoff the posts 308.

During a test, the clamp actuators 284 a-c move the lower clamp 288between three positions: a fully open position, in which each of thethree clamp actuators 284 a-c stalls against the hard stop features 286a-c comprised in the upper clamp 260, and a fully closed position, inwhich at least a portion of the upper clamp 260 abuts at least a portionof the upper clamp 260. In the fully closed position, the cartridge 100is held firmly against the upper clamp 260, thereby engaging thepneumatic and electrical interfaces comprised in the upper clamp withthe corresponding interfaces provided on the exemplary cartridge.Preferably, the cartridge is held against the upper clamp with a forceof at least 100N, more preferably at least 150N and most preferably atleast 200N provided by the clamp actuators 284 a-c, via the lower clamp288.

The movement of the lower clamp relative to the upper clamp throughoutan exemplary test cycle will now be described with reference to FIG. 23,which shows a schematic of the steps involved in an exemplary testcycle.

A user initiates a test cycle by requesting a test. Followinginitiation, the three clamp actuators 284 a-c move the lower clamp 288to the fully open position, defined by a point at which each of thethree clamp actuators 284 a-c stalls against its corresponding hard stop286 a-c. Once all of the clamp actuators 284 a-c have stalled againsthard stops 286 a-c, the lower clamp 288 is aligned parallel to the upperclamp 260 and the isolation valve latching mechanism is primed. Theisolation valve latching mechanism will be described in more detailhereafter.

The lower clamp 288 is then moved to an intermediate ‘home’ position. Inthe home position, the lower clamp 288 is spaced apart from the upperclamp 260, and raised central portion 289 of the lower clamp 288 islocated beneath the cut-out 254 of the drawer 202. When the lower clamp288 reaches the home position, the reader activates the drawerde-latching mechanism described, above the drawer 202 opensautomatically. Once a user has inserted a cartridge 100 and closed thedrawer 202, the lower clamp 288 moves to a fully closed position.

In the fully closed position, the raised central portion 289 of thelower clamp 288 projects through the cut-out 254 in the drawer tray 253,thereby lifting the cartridge 100 out of the drawer 202 and holding init firm contact with the upper clamp 260. This engages the pneumatic andelectrical interfaces on the upper clamp and the exemplary cartridge andaligns the mechanical actuators with their counterparts on the cartridge100.

Once the test has finished, the clamp actuators 284 a-c return the lowerclamp 288 to the home position, lowering the cartridge 100 into thedrawer tray 253 and the drawer 202 opens automatically for the user toremove and dispose of the used cartridge 100.

The clamp actuator system described above is open loop and relies onpositional control of the lower clamp defined by the hard stops 286comprised in the upper clamp 260 for the actuators to stall against.This technique ensures that the lower clamp 288 is always parallel tothe upper clamp 260 at the start of the clamping process and that thefully open position is well defined. However, a skilled person willunderstand that alternative methods of halting the clamp actuators 284a-c may be provided. For example, the motors may be separately actuateduntil the clamp reaches optical sensors (not shown) at the requiredposition.

As shown in FIG. 23 in one embodiment, the exemplary reader 200 may movethe lower clamp 288 to the fully open position after a test has beencompleted and before the lower clamp 288 is returned to its homeposition and the drawer 202 opens for removal of the cartridge. Thisallows the isolation valve latching mechanism described in furtherdetail hereafter to be re-set ready for the next test, before the usedcartridge 100 is removed.

In the exemplary embodiment described above, the upper clamp and thelower clamp are dimensioned such that a distance D between the uppersurface of the lower clamp and the lower surface of the upper clamp isapproximately 0 mm when the lower clamp is in its fully closed position,approximately 7.8 mm when the lower clamp is in its home position andapproximately 14.5 mm when the lower clamp is in its fully openposition. However, a skilled person will understand that distance Dbetween the upper clamp and the lower clamp may be decided based upon anumber of factors. For example, the distance D may be adjusted fordifferent cartridge dimensions, or for different clamp geometries.Moreover, a skilled person will understand that two clamp actuators maybe provided, or four or more clamp actuators.

In the exemplary reader described herein, the upper and lower clamp arepreferably formed of glass fibre reinforced PBT (Polybutyleneterephthalate), each reinforced with a stainless steel bracket. However,a skilled person will understand that other materials may be used forthe upper and lower clamps. For example, PPS (Polyphenylene sulfide) ora PC (polycarbonate) may be used.

2.4 Isolation Valve Actuator

FIG. 24 shows the isolation valve actuation mechanism in more detail. Asshown in FIG. 24, the upper clamp comprises an actuation assembly 310.The actuation assembly 310 comprises an armature 312, which may beformed of a ferromagnetic material, comprised above the upper clamp 260and a second portion 316 comprised below the upper clamp 260, in thecartridge receiving region 271 of the upper clamp 260. A shaft 314passes through an opening 318 provided in the upper clamp 260 andconnects the armature 312 and the second portion 316. A plunger 320,mounted on the second portion 316 aligns with a recess provided in theexemplary cartridge 100, in which the isolation valve 50 is located. Theplunger may comprise any shape, but preferably comprises a flat endportion configured to contact a valve actuator of the isolation valve.

An actuation spring 322 is confined between an upper surface of thesecond portion 316 and a bearing surface 324 provided on the upper clamp260, such that the actuation assembly 310 is biased downwardly withrespect to the upper clamp 260 and into a position in which the plunger320 actuates the isolation valve 50 on the exemplary cartridge 100. Anelectromagnet 326 is mounted on the gantry 298 above the upper clamp, inalignment with the first portion of the actuation assembly 290 below.Preferably, a mounting spring 328 connects the electromagnet 326 to thegantry 298.

The isolation valve activation system shown in FIG. 24 is designed toclose the isolation valve 50 on the cartridge 100 to reduce the escapeof amplified test material. It closes the valve either at the end of thetest or event of a power failure.

At the start of the test, the isolation valve activation mechanism isprimed by the movement of the lower clamp 288 to its fully openposition.

As the lower clamp 288 is moved to the fully open position at the startof the test process, the clamp actuators 284 a-c compress and storeenergy in the two priming springs 300 a-b provided around the twoalignment shafts 294. The springs 300 a-b, are compressed between theupper clamp 260 and the gantry 298 affixed to the lower clamp 288 by thealignment shafts 294. As the lower clamp 260 is moved to its fully openposition, the gantry 298 is lowered to a point at which theelectromagnet 326, attached to the gantry 298, bottoms out on thearmature 312 of the actuation assembly 310. If the electromagnet 326 isspring mounted on the gantry 298, the electromagnet will contact thearmature 312 before the lower clamp reaches its fully open position. Asthe lower clamp 288 moves to the fully open position, the mountingspring 328 is compressed.

The electromagnet 326 is activated to engage the armature 312, such thatit pulls the actuation assembly 310 up, compressing the actuation spring322 when the lower clamp 288 is raised to its home position. When theisolation valve activation mechanism is primed, and the lower clamp 288is in its home position, the plunger 320 is clear of the recess providedin the exemplary cartridge 100.

Preferably, the vertical force provided by the two priming springs 300a-b exceeds the force required to compress the actuator spring. However,a skilled person will understand that no priming springs may be providedor that the vertical force provided by the two priming springs 300 a-bmay be equal to or less than the force require to compress the actuatorsprings and that the clamp actuators may be configured to provide theforce required to overcome the actuation spring as the lower clamp 288moves to its clamped position.

In the exemplary reader described herein, the electromagnet 326 isactivated when the lower clamp reaches its fully open position. However,a skilled person will understand that the electromagnet 326 may beactivated slightly before or after the lower clamp 288 reaches the fullyopen position. Once the isolation valve actuation mechanism has beenprimed, a cartridge 100 may be inserted into the reader 200, and a testcarried out.

At the end of the test (or in the event of power loss) the reader 200de-energises the electromagnet 326, causing the actuation spring 322 toforce the plunger 320 onto the isolation valve 50 in the cartridge 100below, with enough force to close it. Once the reader has actuated theisolation valve, the valve remains latched closed.

Due to geometry constraints, in the exemplary reader 200, the centralaxis of the plunger 320 is not in-line with the central axis of theelectromagnet 396; however the geometry of the mechanism prevents anyjamming of the plunger 320 by ensuring that the forces acting along theactuation assembly 310 from the electromagnet 396 (during priming) andon the isolation valve 50 (during activation) are within the diameter ofthe actuation spring 322. Clearly, a reader according to the presentinvention may be provided in which the central axes of theelectromagnet, actuation assembly and plunger are substantially thesame.

2.5 Thermal Stacks

Referring again to FIG. 21b , the lower clamp 288 comprises threethermal stacks 287 a-c.

FIG. 25 shows a plan view of the raised central portion 289 of the lowerclamp 288, comprising the three thermal stacks 287 a-c. A first thermalstack 287 a is provided at the front of the raised portion 289, and ispositioned to align with the sample preparation region of an exemplarycartridge inserted into the reader. Second and third thermal stacks 287b-c are provided adjacent each other at the rear of the raised portion289. The second and third thermal stacks 287 b-c are positioned to alignwith a sample processing region (for example one or more PCR chambers)of the exemplary cartridge and a detection region of the cartridgerespectively. Each of the first, second and third thermal stacks 287 a-cis aligned with an opening in the lower clamp 288 (shown in FIG. 21b )and is configured to contact and exchange heat with the fluidic foil inthe region of the cartridge with which it is aligned.

In use, the thermal stacks 287 a-c are used to contact and exchange heatwith the fluidic foil bordering the sample preparation region, thesample processing chambers and the detection chambers, therebycontrolling the temperature of the sample within the sample preparationregion, the sample processing chambers and the detection chambers. FIG.28 shows the regions of an exemplary fluidic cartridge that each of thethermal stacks is configured to contact. The first thermal stack isaligned with and configured to contact the fluidic foil in a firstregion 362, which covers the region of the sample mixing chamber 136 andthe coarse filter 162. The second thermal stack is aligned with andconfigured to contact the fluidic foil in a second region 364 whichcovers the sample processing chambers, for example a PCR chamber 56 a-b.The third thermal stack is configured to align with and contact thefluidic foil in a third region which covers the detection chambers 62a-d. As illustrated in FIG. 25, in an exemplary reader configured forused with the exemplary cartridge described above, the first thermalstack is configured to contact the foil across an area of approximately43 mm×22 mm; the second thermal stack 287 b is configured to contact thefoil across an area of 20 mm×20 mm; and the third thermal stack isconfigured to contact across an area of 28 mm by 20 mm. A distance Dbetween the first thermal stacks and the second and third thermal stacksis approximately 50 mm. A skilled person will understand that thesedimensions correspond to a reader designed for use with an exemplarycartridge and that the size and location of the first second and thermalstacks may be modified accordingly to align with and contact therequired regions of an alternative fluidic cartridge.

Turning now to FIGS. 26a-c , each of the first, second and third thermalstacks 287 a-c comprises a spreader plate 330, a reservoir block 338, aheat sink 340, and a fan 342. A peltier device 332 having a first side334 and a second side 336 is provided between the spreader plate 330 andthe reservoir block 332. The first side 334 of the peltier device 332 isin thermal contact with the spreader plate 330 whilst the second side336 of the peltier device is in thermal contact with the reservoir block338. This peltier device is typically a Marlow device, optimised forhigh power and thermocycling purposes.

One or more temperature sensors (not shown) are mounted in the spreaderplate 330 and the reservoir block 338. Typically, a temperature sensoris embedded in a machined recess provided in each of the spreader plate330 and the reservoir block 338, and set in an epoxy resin. However, askilled person will recognise that more than one temperature sensor maybe provided in each spreader plate 330 and each reservoir block 338.Typically, the reservoir block 338 is formed of aluminium, whilst theheat sink 340 is formed of copper, although a skilled person willrecognise that other thermally conductive materials may be used.

In the exemplary reader, first and third thermal stacks 287 a, 287 c aredesigned to provide a static controlled temperature, whilst the secondstack 287 b is designed to change the surface temperature of the secondheater as quickly as possible. This is achieved by modifying thespreader plate 330 b of the second stack 287 b to minimise its mass.

FIG. 27 shows the spreader plate 330 b of the second thermal stack 287 bin more detail.

The spreader plate 330 b has a first face 356 and a second face (notshown), and is preferably comprised of aluminium. The first face 356comprises a substantially planar central portion 349 for contacting thefluidic foil of the exemplary cartridge whilst the second face comprisesa substantially planar central portion for contacting the first side 334of the peltier device 332. As shown in FIG. 27, the spreader plate 330 bhas a substantially rectangular footprint having a long side 344 alongits length and a short side 346 along its width. Fixation points 348 areprovided at each corner of the spreader plate 330 b. Preferably,fixation points 348 are recessed apertures through which a screw may beinserted.

The spreader plate 330 b has first and second cut-outs 350 a-b extendingalong each of its short sides between adjacent fixation points 348. Atthe periphery of each of its short sides 346, the spreader plate 330 bcomprises at least one area of reduced thickness 351 a-c. In theexemplary embodiment shown in FIG. 27, the spreader plate 330 bcomprises a first area of reduced thickness 351 a along one short side,and two further areas of reduced thickness 351 b-c, separated by a ridge352, on the other. One or more areas of reduced thickness 354 a-b arealso provided extending along each of its long sides between adjacentfixation points 348. Preferably, the areas of reduced thickness 354 a-bextending along each of the long sides are provided as elongatedrecesses extending along each of the long sides, such that a ridge 358bounds the outer edge of the long sides 344. However, a skilled personwill understand that one or more areas of reduced thickness may beprovided extending along each of the long edges of the spreader plate.

Typically, the thickness of the spreader plate 330 b is approximately 3mm, except in regions of reduced thickness where the thickness isapproximately 1 mm. By reducing the thickness of the spreader plate 330in at least one of the regions shown in FIG. 27, the mass of thespreader plate can be reduced, whilst maintaining the rigidity requiredto withstand the clamping forces applied repeatedly throughout multipletest cycles.

The first and third spreader plates 330 a,c are preferably formed ofaluminium and typically have a larger, simpler shape than the PCR sincetheir performance is less critical and as such is not optimised.

Referring again to FIGS. 26a-c , to ensure good thermal contact with thecartridge, each thermal stack is spring mounted with respect to thelower clamp 288. Generally, two springs 360 a-b are provided either sideof each thermal stack 287 a-c, as shown in FIGS. 26a-c . Preferably,each thermal stack 287 a-c is spring mounted in the lower clamp 288 suchthat the upper surface of each thermal stack 287 a-c is approximately 1mm proud of the lower clamp when the springs 360 a-b are uncompressed.

At the start of the test, the thermal stacks 287 a-c are brought intocontact with the fluidic foil on the underside of the cartridge 100inserted into the machine as the lower clamp 288 is moved towards itsfully closed position. As the lower clamp 288 moves to its fully closedposition, the springs 360 a-b on which the thermal stacks 287 a-c aremounted are compressed.

Each of the first, second and third thermal stacks 287 a-c are appliedto the fluidic foil of the cartridge with a different force, dependingon the degree of thermal contact required. Typically, the first thermalstack 287 a is applied to the fluidic foil with a first force, thesecond first thermal stack 287 b with a second force and the thirdthermal stack is applied to the fluidic foil with a third force, whereinthe second force is greater than the first force, and the first force isgreater than the second force. Ideally, the first thermal stack isapplied to the foil with a force of 30N±10N; the second thermal stack isapplied to the foil with a force of 45N±10N; and the third thermal stackis applied to the foil with a force of 25N±10N

The force applied to the fluidic foil by the first, second and thirdthermal stacks 287 a-c may be determined by the stiffness of the springs360 a-b on which each of the thermal stacks 287 a-c is mounted in thelower clamp 288. Alternatively, the distance by which each of the stacksprotrudes may be varied, such that each of the springs 360 a-c must becompressed by a different amount.

The surface temperature of each of the thermal stacks 287 a-c isindividually controlled by the thermal board 221, which is mounteddirectly below the main control board 220. A schematic diagram of thethermal board is shown in FIG. 15. It carries drive circuits for thepeltier devices 232 a-c and the three DC fans. These circuits connect tothe peltier devices and fans via the connector board 224.

In use, the surface temperature of each of the first, second and thirdthermal stacks is monitored by the thermal sensors (not shown) andmaintained by the peltier drivers on the thermal board. The temperaturesensors are configured to measure a temperature of the surface of eachthermal stack 287 a-c. This information is fed back to the thermalboard, which, based on the signal from the temperature sensors at thesurface of each of the thermal stacks 287 a-c, varies the signalsupplied by the peltier driver to each of the peltier devices 232 a-c toachieve a desired temperature at the fluidic foil. As will be understoodby the skilled person, the first side of each peltier device may bedriven as a cooler or a heater for each portion of the fluidic foil asrequired.

During a test, the thermal board is typically configured to maintain thefirst thermal stack at a constant temperature between 35° C. and 40° C.,preferably approximately 37° C.

The design of the second thermal stack, including the low mass of thesecond spreader plate, allows rapid cycling of the sample processingregion. Typically, the second thermal stack cycles the temperature ofthe sample processing region between approximately 65° C. andapproximately 95° C. Due to the low mass of the spreader plate 330 b, acycle time of between 7 and 8.5 s is possible (cycle=time taken for thesurface of the thermal stack to go from 65° C.-95° C., and from 95°C.-65° C.). Preferably, one cycle is 7 s. Typically, 35 to 45(preferably 40) thermal cycles are completed in a single test sequence.Thereafter, the PCR stack is held at a constant temperature for movementof the sample to the detection chamber.

Preferably, the third thermal stack maintains a surface temperature ofapproximately 20° C. This prevents the reagents provided in thedetection chambers 62 a-d from overheating. In practice, since thesample processing region is adjacent to the detection chamber and isheated to much higher temperature, the third thermal stack is configuredas a cooler for the detection chamber for at least a part of the testcycle.

After a test has been completed, the lower clamp returns to its homeposition, and the thermal stacks 287 a-c move out of contact with thefluidic foil once the cartridge has been lowered into the drawer 202. Byproviding the second thermal stack at the rear of the lower clamp, thesecond thermal stack (having the highest temperature) is not accessibleto the user, even when the drawer is open.

2.6 Blister and Valve Actuation

Referring now to FIG. 29, the upper clamp 260 comprises four mechanicalactuators; three mechanical blister actuators 278 a-c and a mechanicalvalve actuator 280. Each of the blister actuators comprises a housing395, a linear actuator (not shown), a stem portion 395, and a tipportion 370. Each of the linear actuators preferably comprises a steppermotor engaged with a splined shaft (for example a Haydon Kerk unit,model no. 25443-05-048) although a skilled person will understand thatother linear actuators may be provided.

In the exemplary cartridge, three blisters are provided, containing oneeach of lysis buffer, wash buffer, and elution buffer. However, askilled person will recognise that the blister actuators describedherein may be implemented with any fluid-filled blister sub-assembly.

FIG. 30 shows the tip geometries of the first, second and third blisteractuators 278 a-c each comprising stem 395 and a tip 370. Each tipcomprises a diameter D, and is dimensioned to align with and collapse acorresponding blister on a cartridge 100 inserted between the upper andlower clamps. Preferably, the actuator tip configured to actuate thelysis blister of the exemplary cartridge has a diameter D_(L) ofapproximately 19.5 mm; the blister tip configured to actuate the washblister has a diameter D_(W) of 15.0 mm; and the actuator tip configuredto actuate the elution blister has a diameter D_(E) has a diameter ofapproximately 10.1 mm, although a skilled person will understand thatthe radius of each actuator tip may be modified reflect the size of theblister to be actuated. A spring 374 (shown in FIG. 20) may be providedbetween the actuator housing and the blister actuator tips 370.

As shown in FIG. 30, the actuator tip may have a domed actuation surfaceconfigured to contact a collapsible blister comprised in the exemplarycartridge. Ideally, the radius of curvature R of the domed surface forthe lysis actuator is 17.3 mm, the radius of curvature for the washactuator is 15.1 mm, and the radius of curvature for the elutionactuator is 11.5 mm. In one embodiment, at least one of the first,second and third blister actuators comprise a flat tip for contactingthe corresponding blister. By providing a blister actuator having asubstantially flat tip, the reader can expel substantially all of thefluid contained in the collapsible blister.

In use, the reader is configured to actuate the lysis blister, then thewash blister and then the elution blister. In order to actuate eachblister, the reader moves the actuator from a first position in whichthe actuator tip is spaced apart from the blister beneath it, to asecond position, in which the blister actuator has entirely collapsedthe collapsible blister and expelled the contents into the cartridge.Preferably, the reader holds the blisters actuators 278 a-c in theirsecond position for a time T after the blister has been collapsed,thereby preventing the expelled contents of the blister from re-enteringthe blister chamber. Preferably, time T is at least 5 s, more preferablyat least 7 s and more preferably approximately 10 s. Preferably, thedistance travelled by each actuator tip is at least 30 mm, morepreferably at least 40 mm and more preferably approximately 44 mm.Preferably, the reader maintains the each blister actuator in its fullyactuated position. Furthermore, each of the linear actuators areconfigured so that it is impossible to back-drive the tips, therebypreventing the blister contents re-entering the blister after actuation.An example of a suitable linear actuator that cannot be back-driven maybe a Haydon Kerk unit, model no. 25443-05-048.

Referring now to FIGS. 31a-b , the upper clamp further comprises amechanical valve actuator 280. The mechanical valve actuator 280 alignswith and is configured to actuate a mechanical valve comprised in acartridge 100 inserted into the reader.

As shown in FIG. 31b , the mechanical valve comprised in the exemplarycartridge preferably comprises a valve chamber 400, containing first andsecond valve seats. The first valve seat comprises a first opening 402,whilst the second valve seat comprises second and third openings 403,404. The first opening is spaced apart from the second opening by adistance a, and the second opening is spaced apart from the thirdopening by a distance b, wherein a>b. A valve membrane seals the valvechamber, and comprises a first portion, for sealing against firstopening in the first valve seat, and a second portion, for sealingagainst the second and third openings in the second valve seat.

FIG. 31a shows the mechanical valve actuator 280 in more detail. Themechanical valve actuator comprises a first actuation portion 376, asecond actuation portion 378. The first actuation portion comprises asubstantially oblong body having a first substantially planar portion380 for contacting the first valve membrane portion. The substantiallyplanar portion 380 comprises a length L and a width W, wherein thelength is longer than the width. Typically, the first actuation portion376 is dimensioned to fit within the valve cavity comprised in theexemplary cartridge, and has a complementary footprint, with roundedcorners, which covers substantially all of the valve membrane. Thesecond actuator portion 378 comprises a substantially cylindrical body,having a second substantially planar portion 382 for contacting thesecond valve membrane portion, and is provided on the first planarportion 380 of the first actuation member. A diameter D of the secondportion is equal to or less than the width W. Preferably D=W=7 mm. Thesecond actuator portion 378 is provided towards one end of the length L,and is movably mounted relative to the first actuator portion 376 andbiased by a spring 277 away from the first portion, towards a cartridgeclamped between the upper and lower clamps. The substantially oblongbody of the first actuation portion comprises a recess into which thesecond actuation portion fits such that when the spring 277 iscompressed, the lower surfaces of the first and second actuationportions are flush. Optionally, fixation means 406 a-b may be providedon each of the long sides.

The reader is configured to actuate the mechanical valve in two stages,of a continuous movement, the first actuator portion sealing the firstportion of the valve membrane against the first opening, whilst thesecond portion of the actuator seals the second portion of the valvemembrane against the second and third openings.

In use, the reader closes the mechanical valve by sealing the second andthird openings, then sealing the first opening. The reverse is true whenthe valve opens—the first valve opening is opened, then the second andthird valve openings are opened.

To close the valve, the reader moves the first actuator portion towardsa clamped cartridge, thereby bringing the second actuator portion intocontact with the second portion of the valve membrane. The secondactuator portion deforms the second valve membrane portion, and seals itagainst the second and third openings. At this point, the first actuatorportion is spaced apart from the first valve membrane portion.

As the actuator continues to move the first actuator towards thecartridge the spring 377 is compressed between the second actuatorportion and the first actuator portion. As the first actuator portioncontinues to move towards the clamped cartridge, the first actuatorportion contacts the first portion of the valve membrane, eventuallydeforming the valve membrane and sealing it against the first opening.

To open the valve, the reader moves the first actuator portion towardsthe upper clamp, away from the clamped cartridge. In doing so, the firstactuator portion moves to a position in which it no longer seals thefirst valve membrane portion against the first opening, whilst thesecond actuator portion maintains the seal over the second and thirdopenings under the influence of the spring 377. As the first actuatorportion moves further from the cartridge, the spring 377 reaches itsfull extension and the second actuator portion is moved away from thesecond valve membrane portion, thereby opening the second and thirdopenings.

The mechanical valve actuator 280 may also be used in combination withthe isolation valve actuator to depressurise the back end of thecartridge once a test has been completed. In order to depressurise theback end of the cartridge, the reader seals the first portion of thevalve membrane against the first and second valve seats, and then sealsthe second valve membrane portion against the third valve seat. At theend of the test, reader actuates the isolation valve actuator, andcloses the isolation valve in the fluidic cartridge as described above.In closing the isolation valve, the reader creates a closed systemwithin the back end of the cartridge. Following actuation of theisolation valve, the actuation valve is opened, as described above,thereby increasing the volume of the closed system sealed by theisolation valve.

2.7 Pneumatics

The pneumatics system will now be described with reference to FIGS. 32to 38. The system comprises a pneumatics block situated within thecartridge reader housing, connected to a pneumatics manifold whichincludes eleven pneumatic ports that form a pneumatics interface thatconnects to a pneumatics interface on a cartridge inserted into thereader.

FIGS. 32 to 37 show the pneumatics block 410, which is constructed frommachined aluminium and associated components. The pneumatics blockcomprises all components necessary to generate a positive pressure and anegative pressure for use in controlling bellows and valves using‘pneumatic logic’ (explained further below), a reference pressure, or apositive ‘blower’ pressure for evacuating a channel in the cartridge.These components include, among others, a pump for generating positivepressure (henceforth a pressure pump); a pump for generating negativepressure (henceforth a vacuum pump); three pressure reservoirs—twopositive pressure reservoirs and one negative pressure reservoir; and aplurality of solenoid valves, each of which couples one pneumatic portto one or more pressure reservoirs.

Provided on the pneumatic block are eleven pneumatics ports 41 1 whichconnect to pneumatic tubes, which in turn connect to the pneumaticsmanifold. FIG. 35 show an interface 412 on the manifold to which thepneumatic pipes from the pneumatic block are connected. FIG. 36 shows aninterface 413 a on the manifold which is configured to couple to acorresponding interface 413 b on the cartridge.

As shown in FIG. 36, the pneumatic ports on the pneumatics manifold arearranged in two rows of 5 ports and 4 ports, respectively. The ports areoffset from each other such that a port in one of the rows is alignedequidistantly between two ports of the other. The ports are numbered,and port number 1 is located at one end of the row of 5 ports. Eachsubsequent port number is adjacent the previous port but on the otherrow. Thus, the row of 5 ports contains odd-numbered ports (numbers 1, 3,5, 7, 9 and 11); whereas the row of 4 ports contains even-numbered ports(numbers 2, 4, 6, 8 and 10). The numbering of the ports will beexplained in more detail below.

FIG. 37 shows the pneumatic interface located on the upper clamp.

FIG. 38 shows a pneumatic circuit diagram for the pneumatic block. Thecircuit comprises a positive pressure sub-system and a negative pressuresub-system. Each of the eleven pneumatic ports is coupled to a solenoidvalve, of which eight are ‘3-port; 2-position’ solenoid valves that arecoupled to supply lines of the positive pressure sub-system and thenegative pressure sub-system (henceforth, the ‘pneumatic logic’ solenoidvalves); of which a further two are 2-port; 2-position′ solenoid valvesthat are coupled to a supply line of the positive pressure sub-systemonly (henceforth, the ‘blower’ solenoid valves); and a final one is a‘3-port; 2-position’ solenoid valves that is coupled to a supply line ofthe positive pressure sub-system only (henceforth the‘reference-pressure’ solenoid valve).

The negative pressure subsystem comprises a vacuum pump coupled to avacuum reservoir. The pump is configured to maintain a gauge pressure of−0.5 barg in the vacuum reservoir. The vacuum reservoir is coupled to a‘2-port; 2-position’ solenoid valve which controls whether or not vacuummay be applied to a supply line to which the eight ‘pneumatic logic’solenoid valves are each coupled.

The positive pressure subsystem comprises a pressure pump coupled tofirst and second pressure reservoirs. The pump is configured to maintaina gauge pressure of 1 barg in the first pressure reservoir and tomaintain a gauge pressure of 1.5 barg in the second pressure reservoir.A ‘2-port; 2-position’ solenoid valve selectively couples the pump tothe first and second pressure reservoirs. The first pressure reservoirapplies pressure to a first supply line to which the eight ‘pneumaticlogic’ solenoid valves are each coupled.

The eight ‘pneumatic logic’ solenoid valves each have an output portcoupled to a pneumatic interface port, and first and second input ports.By default (i.e. in their normal, unactuated positions) the valves areconfigured to couple the first input port to the output port, and mustbe actuated in order to couple the second input port to the output port.The supply line of the negative pressure sub-assembly is coupled to thefirst input ports of the eight ‘pneumatic logic’ solenoid valves,whereas the first supply line of the positive pressure sub-assembly iscoupled to the second input ports. Thus, by default (and assuming the‘2-port; 2-position’ solenoid valve of the negative pressuresub-assembly is open), the eight ‘pneumatic logic’ solenoid valves allowa negative pressure to be applied to their corresponding pneumaticinterface ports. When individually or collectively actuated, the eight‘pneumatic logic’ solenoid valves allow a positive pressure to beapplied to their corresponding pneumatic interface ports.

The second pressure reservoir applies pressure to a second supply lineto which the two ‘blower’ solenoid valves and the ‘reference-pressure’solenoid valve are each coupled. More specifically, the second supplyline branches into first a second branches, the first of which iscoupled to the two ‘blower’ solenoid valves. The second branch of thesecond supply line is coupled to the ‘reference-pressure’ solenoid valvevia a pressure regulator.

The two ‘blower’ solenoid valves each have an output port coupled to apneumatic interface port, and an input port coupled to the secondpressure reservoir. By default (i.e. in their normal, unactuatedpositions) the valves are closed, such that the input port isdisconnected from the output port. When actuated, the two ‘blower’solenoid valves allow a positive pressure to be applied to theircorresponding pneumatic interface ports. The second pressure reservoiris configured to supply a flow rate of at least 1.5 litres per minute at1 barg for this purpose.

The ‘reference-pressure’ solenoid valve has an output port coupled to apneumatic interface port, and first and second input ports. By default(i.e. in their normal, unactuated positions) the valves are configuredto couple the first input port to the output port, and must be actuatedin order to couple the second input port to the output port. The firstinput port is a channel connected to a further ‘vent’ solenoid valvethat is normally closed but nay be opened to atmosphere, whereas thesecond input port is coupled to the second pressure reservoir via thepressure regulator. Thus, by default, the ‘reference pressure’ solenoidvalve seals the corresponding pneumatic interface port. When the ‘vent’solenoid is actuated, the pneumatic interface is connected toatmosphere. When the ‘reference pressure’ solenoid valve is actuated, itallows a regulated pressure to be applied to the corresponding pneumaticinterface port.

A controller on the main board controls the actuation of the pressurepump, the vacuum pump and all solenoid valves. Thus, during a test, thecontroller may operate the pumps and valves according to a predeterminedcycle.

The pneumatics system performs 4 basic functions on a fluidic cartridge.The first is to operate the bellows that pumps the fluid sample aroundthe cartridge; the second is to operate the various pneumatic valveslocated in the cartridge; the third is to evacuate channels in thecartridge; and the fourth is to provide a reference pressure.

Operation of the bellows and valves is performed by the eight ‘pneumaticlogic’ solenoid valves. The valves are capable of applying either apositive pressure (of 1 barg) or a negative pressure (of −0.5 barg) toeach pneumatic port coupled to those valves. The valves and bellows ofthe cartridge are open when a negative pressure is applied and closedwhen a positive pressure is applied. Thus, the bellows and pneumaticvalves of a cartridge may be controlled.

The operation of the bellows will be described by way of example. Avalve is placed either side of the bellows. Air is drawn in to thebellows by opening the bellows (applying a negative pressure) whilst theupstream valve is open (applying a negative pressure) and the downstreamvalve is closed (applying a positive pressure). Then, the upstream valveis closed (applying a positive pressure) and air is expelled from thebellows by closing the bellows (applying a positive pressure) whilst thedownstream valve is open (applying a negative pressure). This cyclerepeats to pump a fluid downstream.

Evacuation of channels in the cartridge is performed by the two ‘blower’solenoid valves. These valves are capable of applying a positivepressure of 1.5 barg, and achieve a flow rate of at least 1.5 litres perminute at a pressure of 1 barg. Each valve evacuates and dries adifferent section of channels of the cartridge.

It will be appreciated that in order to evacuate and dry the channels ofthe cartridge, it is necessary to open pneumatic valves. Since thereader is able to apply a negative pressure and a positive pressuresimultaneously (owing to the two pumps), it is possible to open therequisite pneumatic valves on the cartridge by operating the appropriate‘pneumatic logic’ solenoid valves accordingly, and then evacuate thechannels of the cartridge by operating the ‘blower’ solenoid valvesaccordingly.

In the preferred case (corresponding to the exemplary cartridgedescribed above), pneumatic interface port numbers 1 to 4, 6, 8, 10 and1 1 are coupled to ‘pneumatic logic’ solenoid valves; pneumaticinterface port numbers 5 and 7 are coupled to ‘blower’ solenoid valvesand pneumatic interface port number 9 is coupled to thereference-pressure solenoid valve.

Between each of the two ‘blower’ solenoid valves and the‘reference-pressure’ solenoid valves and their respective pneumaticinterface ports there is provided a fluid trap for capturing any fluidthat flows from the cartridge into the reader, thus preventing suchfluid from damaging the reader.

Each pneumatic interface port shall be not less than 3 mm in diameter,and the pneumatic interface of the exemplary cartridge shall beconfigured to receive pneumatic interface ports that are not less than 3mm in diameter.

2.8 Improved Arrangement of Electrodes and Cartridge Reader

As discussed above, the reader comprises an electrical interface,comprised in the upper clamp and configured to contact the electrodescomprised in the cartridge.

Conventionally, the counter electrode in a potentiostat is larger thanthe working electrode to provide an ample supply of surplus electrons.However, it has been found that reversing this convention surprisinglyoffers better results for the exemplary cartridge. For theelectrochemistry performed on the liquid sample described above in theexemplary cartridge, it is found that having a working electrode whichis larger than the counter electrode provides larger signals andimproved results by way of increased sensitivity. In other words, havinga current flow from a relatively large working electrode to a relativelysmall counter electrode offers improvements over the conventionalarrangement. The improvement is particularly effective for detectinglabels comprising modified Ferrocene Carboxylic Acids in aqueousbuffers.

Referring to FIG. 10, preferably each working electrodes 169 a-d isformed in a U-shape and each counter electrode 170 a-d is formed in astraight elongate shape between the two prongs of the respectiveU-shaped working electrode.

In FIG. 10, each of the working electrodes 169 a-d has a total length of8.1 nm Each electrode is made up of a short prong 174 a-d of length 5.1mm, connected to an interconnecting portion 176 a-d of length 1.3 mmoriented at 90° to the short prong 174 a-d, connected to a long prong178 a-d of length 8.1 mm oriented at 90° to the interconnecting portion176 a-d and parallel to and adjacent with the short prong 174 a-d. Thewidth of the working electrodes is 0.74 mm along its length, whichprovides a total surface area of 10.2 mm².

Each of the counter electrodes 170 a-d is made up of an elongate portionof length 7.2 mm and width 0.35 mm, which provides a total surface areaof 2.5 mm. Thus, the ratio of surface area of the counter electrode tothe surface area of the working electrode is 1:4. This ratio has beenfound to be particularly effective at providing larger signals that canbe measured effectively because of the increased sensitivity.

As shown in FIG. 10, each electrode in the set of electrodes isconnected to an electrical contact for electrically connecting to acorresponding electrical contact in a cartridge reader. For instance,each working electrode 169 a-d is connected to an electrical contact 180a-d; each counter electrode 170 a-d is connected to an electricalcontact 184 a-d; and each reference electrode 171 a-d is connected to anelectrical contact 182 a-d. For a cartridge having four detectionchambers, there are 12 electrical contacts 180 a-d, 182 a-d, 184 a-d.

FIG. 39 shows a portion of the housing of the exemplary cartridge inwhich the invention is implemented. Visible in the figure is theelectrical interface 102 shown in FIG. 39. The electrical interfacecomprises the 12 electrical contacts 180 a-d, 182 a-d, 184 a-d which areaccessible through an elongate aperture 186 in the housing, therebyproviding a contact area on each electrical contact 180 a-d, 182 a-d,184 a-d for electrically connecting to a corresponding electricalcontact on the reader. As shown, each electrical contact presents acontact area of 2.7 mm by 1.5 mm. It is possible to provide smaller orlarger contact areas for each electrical contact, for instance thecontact areas may each be at least 0.5 mm by 0.5 mm; preferably at least1 mm by 0.5 mm; more preferably at least 2 mm by 1 mm, more preferablyat least 2.7 mm by 1.5 mm.

A reader into which the exemplary cartridge is inserted in order toperform a test comprises a control module and an electronics interface(not shown) having 12 electrical contacts corresponding to the 12electrical contacts on the cartridge. The electrical contacts of thereader take the form of 12 pins which are sprung so as to be biasedtoward a cartridge inserted into the reader. When a cartridge isinserted into the reader and the pins contact the electrical contacts ofthe cartridge, the pins apply a spring force of between 300 and 900 mN(per pin) against the electrical contacts. The pins are gold plated toprovide optical electrical connectivity between the cartridge and thereader.

To carry out an electrochemical test on a sample in the cartridge, thecontrol module and electronic interface of the reader apply a potentialdifference across the pins which contact the counter, references andworking electrodes of the cartridge (180 a-d; 184 a-d). The currentflowing between these electrodes is then measured. A suitable circuitfor applying this potential difference and measuring the resultingcurrent is shown in FIG. 10, together with a diagrammatic representationof what is taking place in the circuit. In summary, a signal generatorin the control module and electronic interface of the reader applies apotential difference (V_(applied)), corrected for activity on thereference electrode, across the working and counter electrodes, and theresulting current flowing from the working electrode to the counterelectrode is measured by a current measuring means I. In a given testcarried out by the reader, the potential difference applied across thepins which contact the counter electrodes and working electrodes of thecartridge (180 a-d; 184 a-d) varies according to a voltage sweep, asdescribed below.

The pins in the reader which contact the reference electrodes of thecartridge (182 a-d) are coupled to a voltage measuring means V. Thecircuit shown in FIG. 40 is thus able to measure the voltage between thereference electrode and the working electrode (V_(contro)l) and thevoltage between the reference electrode and the counter electrode(V_(reference)). The reference electrode is used as a reference suchthat all other voltages are declared with respect to this referenceelectrode, and therefore pertain to the activity of the workingelectrode only. This is because the voltage of the reference electrodeis largely independent of the redox conditions and therefore means thatit is only the redox state of the chemistry at the working electrodethat is being measured.

The control module and electronic interface of the reader are configuredto conduct a test using differential pulse voltammetry or square wavepulse voltammetry.

A first exemplary test using differential pulse voltammetry will now bedescribed with reference to FIG. 41.

In the first exemplary test, a slowly incrementing voltage sweep isapplied across the working and control electrodes (i.e. V_(applied))with a relatively large step pulse overlaid at the start of eachincrement. The full sweep is from around −0.7 volts to +1 volts relativeto reference electrode. This range is within the practical half-cellranges limited by partial hydrolysis of water and degradation of DNAmolecules themselves.

Each pulse has a magnitude of 50 mV and the sweep increases by a stepincrement of 3 mV per pulse. The duration of the test may be shortenedby increasing the step increment, but this leads to coarser results.Accordingly, a step increment of between 1 mV and 5 mV is preferred,preferably between 2 mV and 4 mV.

Each pulse causes two different currents to flow through the sample—oneduring the peak of the pulse and one during the trough of the pulse. Thecurrent flowing during the pulse peak is the forward current (M1) andthe current flowing during the pulse trough is the reverse current (M2).These currents are measured, and may be plotted against V_(applied)(relative to V_(reference)) In particular, the differential currentbetween M1 and M2 may be plotted against V_(applied) (relative toV_(reference)), as shown in FIG. 42. From these, a peak differentialcurrent associated with electrochemical activity of a label may beidentified, providing the signal that is indicative of the labels in thesample.

The skilled person will be capable of modifying the exemplary cartridgeto implement the inventive aspects described herein in various waysdepending on circumstances. It is intended that the scope of the presentinvention is defined by the following claims.

1-9. (canceled)
 10. A cartridge reader for carrying out a diagnostictest on a fluid sample contained in a fluidic cartridge in the cartridgereader, the fluidic cartridge comprising bellows for moving the fluidsample along a network of channels, a plurality of pneumaticallyactuated valves in the network of channels; and a pneumatics interface,the cartridge reader comprising: a pneumatics system, comprising: apneumatics interface, comprising a plurality of pneumatic interfaceports, configured to couple to the pneumatics interface on the fluidiccartridge; a positive pressure sub-system comprising first and secondpositive pressure reservoirs and a first pump configured to provide asupply of positive pressure to maintain the first and second positivepressure reservoirs at first and second positive pressures,respectively, wherein the first and second positive pressures aredifferent; and a negative pressure sub-system comprising a negativepressure reservoir and a second pump configured to provide a supply ofnegative pressure to maintain the negative pressure reservoir at anegative pressure; a valve system comprising a plurality of solenoidvalves, each connected to a pneumatic interface port and configured tocouple to that pneumatic interface port at least one of: the first andsecond positive pressure reservoirs and the negative pressure reservoir;and processing means configured to operate the first and second pumpsand the plurality of solenoid valves according to a predetermined cycleduring the diagnostic test such that during the cycle: a first subset ofpneumatic interface ports is selectively coupled to either the firstpositive pressure reservoir or the negative pressure reservoir foractuating the bellows and the pneumatic valves on the cartridge; and asecond subset of pneumatic interface ports is coupled to the secondpositive pressure reservoir for evacuating a surplus fluid from at leasta portion of the network of channels in the fluidic cartridge.
 11. Thecartridge reader of claim 10, wherein the positive pressure sub-systemfurther comprises a pressure regulator connected between the secondpositive pressure reservoir and a reference-pressure solenoid valve, andwherein the processing means is configured to operate thereference-pressure solenoid valve such that a reference-pressurepneumatic interface port is coupled to the second positive pressurereservoir via the pressure regulator for providing a reference pressureto the fluidic cartridge.
 12. The cartridge reader of claim 10, whereinthe first positive pressure is a positive gauge pressure of 1 bar. 13.The cartridge reader of claim 10, wherein the negative pressure is anegative gauge pressure of 0.5 bar.
 14. The cartridge reader of claim11, wherein the reference pressure is a gauge pressure of between 0 and1 bar and the pressure regulator is configured to maintain the referencepressure at 0.5% full scale accuracy.
 15. The cartridge reader of claim10, wherein the positive pressure sub-system further comprises one ormore fluid traps between a corresponding one or more pneumatic interfaceports and their respective solenoid valves.
 16. The cartridge reader ofclaim 15, wherein the one or more fluid traps includes a fluid trapbetween each of the second subset of pneumatic interface ports and theirrespective solenoid valves.
 17. The cartridge reader of claim 15,wherein the one or more fluid traps includes a fluid trap between thereference-pressure solenoid valve and the reference-pressure pneumaticinterface port.
 18. The cartridge reader of claim 10, wherein thepneumatic interface comprises eleven pneumatic interface ports alignedin first and second rows, consisting of five pneumatic interface portsand four pneumatic interface ports, respectively, wherein the pneumaticinterface ports of the first and second rows are offset from each other.19. The cartridge reader of claim 18, wherein pneumatic interface portnumber 1 is situated at one end of the first row, and each subsequentlynumbered port is adjacent the preceding port of the opposite row. 20.The cartridge reader of claim 19, wherein the first subset of pneumaticinterface ports consists of port numbers 1, 2, 3, 4, 6, 8, 10 and 11.21. The cartridge reader of claim 20, wherein the second subset ofpneumatic interface ports consists of port numbers 5 and
 7. 22. Thecartridge reader of claim 19, wherein the reference-pressure pneumaticinterface port is port number
 9. 23. The cartridge reader of claim 10,wherein the positive pressure sub-system, negative pressure sub-systemand valve system and provided on a pneumatics block, and the pneumaticsinterface ports are provided on a pneumatics interface manifold that isconnected to the pneumatics block by a plurality of pneumatics pipes.24. The cartridge reader of claim 23, wherein the reader comprises afixed upper clamp and a movable lower clamp configured to hold acartridge within the cartridge reader, and wherein the pneumaticsinterface manifold is mounted to the upper clamp by one or more springs.25. The cartridge reader of claim 24, wherein the one or more springsare configured to apply a biasing force of between 30 and 60N,preferably between 40 and SON, preferably 45N. 26-27. (canceled)